ES2952048T3 - New peptides that have antimicrobial activity and new enzyme capable of converting an L-configuration residue to a D-configuration amino acid in a peptide - Google Patents

Abstract

The present invention relates to a new class of peptides that have antibacterial activity and are present in D-amino acids and their uses. It also refers to a new enzyme that presents peptide epimer activity in vitro and in vivo, being useful for modifying peptides in order to change the amino acid configuration from L to D. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

New peptides that have antimicrobial activity and new enzyme capable of converting an L-configuration residue into a D-configuration amino acid in a peptide

FIELD OF THE INVENTION

The present description relates to medicine, in particular to the antimicrobial activity of peptides and to enzymology.

BACKGROUND OF THE INVENTION

Antimicrobial resistance threatens the effective prevention and treatment of an increasing range of infections caused by bacteria, parasites, viruses and fungi. It is an increasingly serious threat to global public health that requires action across all sectors of government and society. Antimicrobial resistance is present in all parts of the world. New resistance mechanisms emerge and spread globally.

Considering the continuing problems due to antimicrobial resistance, there is a strong need for new classes of antimicrobial agents. In particular, antimicrobial peptides are of great interest. These peptides are generally potent. Furthermore, they often offer better selectivity and specificity than the small molecules generally used as therapeutic agents.

However, an important limitation to its use and therapeutic development is associated with its half-life. In fact, it usually decreases due to the action of proteases and peptidases that are present in organisms.

Therefore, numerous strategies have been developed in order to reduce peptide degradation, such as C-amidation and N-acetylation against exopeptidase, the introduction of unnatural amino acids or D-configuration amino acids. However, these strategies only They can be used in the context of synthesized peptides and are not suitable for recombinant peptide production. In particular, there is no easy way to introduce amino acids with a D configuration into a peptide, despite the interest of such a modification. In fact, the D-configuration amino acid has the same properties as natural amino acids, but is generally more resistant to proteases and could even be less immunogenic.

In conclusion, any new class of antimicrobial agents is of great interest, especially antimicrobial peptides. Furthermore, any suitable procedure for modifying L-configuration amino acids to D-configuration amino acids in a peptide would be very valuable in this context.

Butcher et al. (2007, J. Bacterio!., 189, 8676) identified a YydFGHIJ operon that appeared to induce LiaRS expression. The amino acid sequences of a yydF peptide are described in Uniprot:Q45596. Feitelson et al. (2006, Cancer Letters, 239, 10-20) refer to epigenetic and genetic changes.

SUMMARY OF THE INVENTION

The scope of the present invention is defined by the claims and any information not included in the claims is provided for informational purposes only.

The inventors identified an antimicrobial peptide derived from bacteria, namely from Bacillus subtilis possessing D amino acid residues. Therefore, the inventors have discovered a new class of bacteriocins. This peptide has the specificity to present the antimicrobial effect only when some of its amino acids are in the D configuration. This peptide can be prepared using an enzyme derived from the same bacteria that has the property of changing the configuration of the amino acids in a peptide from from an L configuration to a D configuration. Therefore, this enzyme may be useful for the conversion of L configuration amino acids to D configuration amino acids in peptides, particularly those prepared by recombinant production.

Accordingly, the present description relates to an antimicrobial peptide having at least two amino acids with D configuration, comprising a sequence of 17 residues having at least 40% identity with the sequence at positions 33 to 49 of SEQ ID No. 1 and said sequence comprising the sequences WYF-[V/I/A] (SEQ ID NO. 71) and W-[l N /A]-X4-GS (SEQ ID NO. 69), in wherein Nos. 69 and 71 have a D configuration.

Preferably, the peptide comprises, essentially consists of or consists of a sequence of WYF-[V/I/A]-[K/R]-Xa-Xb-Xc-NRW-[V/I/A]-Xd-GS -Xe-H (SEQ ID NO: 72), wherein Xa, Xb and Xc are a polar amino acid, Xd is an aliphatic amino acid and Xe is any amino acid. More preferably, the peptide comprises, essentially consists of or consists of a sequence of WYF-[V/I/A]-[K/R]-[S/N]-[S/Q/K]-[E/K /S/Q]-NRW-[V/I/A]-[L N /A]-GS-[ N G]-H (SEQ ID NO: 73).

In a preferred aspect, the peptide comprises, essentially consists of or consists of a sequence selected from the group consisting of SEQ ID NO:61-68

In one aspect, the peptide comprises a 24-residue sequence having at least 40% identity with the sequence at positions 26 to 49 of SEQ ID NO: 1 and said sequence comprising the sequences N/DDLWYF-[V/ I/A] (SEQ ID NO: 21) and W-[l N /A]-X4-GS (SEQ ID NO: 69), wherein X4 is any amino acid and a linker sequence between SEQ ID NO: #21 and SEQ ID #69 of 4-7 amino acids.

Preferably, the peptide comprises the sequences of L-X1-X2-X3-NDLWYFV/I (SEQ ID NO: 23) and WI N -X4-GS (SEQ ID NO: 22), wherein X1, X2 , X3 and X4 are any amino acid. More preferably, X1 is alanine, aspartic acid or glutamic acid, X2 is lysine, asparagine or aspartic acid and X3 is valine, isoleucine or glutamine.

Preferably, the peptide comprises the sequences LAKVNDLWYFV (SEQ ID NO: 24) and Wl N -X4-GS (SEQ ID NO: 22), wherein X4 is a hydrophobic amino acid, preferably leucine, valine, alanine or methionine.

Preferably, the valine or isoleucine in the sequence N/DDLWYF- [V/I/A] (SEQ ID NO: 21) has a D configuration and the isoleucine or valine in the sequence W- I N -X4-GS ( SEQ ID NO: 22) has a D configuration.

In a preferred aspect, the peptide comprises, consists essentially of or consists of the sequence of SEQ ID NO: 20 and the valine at position 19 is in D configuration and the isoleucine at position 27 is in D configuration.

The present description also relates to a pharmaceutical composition comprising a peptide according to the present description.

The present description further relates to a peptide according to the present description for use as a medicament, preferably as an antimicrobial, more preferably as an antibacterial. It refers to the use of a peptide according to the present description for the manufacture of a medicament, preferably an antimicrobial agent, more preferably an antibacterial. The present description relates to a method of treating a subject in need thereof, comprising administering a therapeutic amount of a peptide according to the present description.

The present description relates to an in vitro method for preparing a peptide according to the present description, wherein the method comprises a step of contacting a peptide comprising a sequence of 17 residues having at least 40% identity with the sequence at positions 33 to 49 of SEQ ID NO: 1 and said sequence comprising the sequences WYF-[V/I/A] (SEQ ID NO: 71) and W-[l N /A] -X4-GS (SEQ ID NO: 69), where X4 is any amino acid and a junction sequence between SEQ ID NO: 69 and SEQ ID NO: 71 of 4-8 amino acids, with an epimerase of the radical SAM peptide having an amino acid sequence that has at least 30% identity with SEQ ID NO: 25 and in which 70% of positions 14-15, 18, 20-24, 27, 58 -60, 63, 83, 84, 87-90, 112, 115, 117, 120, 150, 152, 169, 176, 180, 181, 183, 204, 206-208, 214, 217, 220-225, 228 , 230, 252, 254, 262, 289, 292, 296 and 309 of SEQ ID NO: 25 are retained.

In one aspect, the peptide comprises a 24-residue sequence having at least 40% identity with the sequence at positions 26 to 49 of SEQ ID NO: 1 and said sequence comprising the sequences N/DDLWYF-[V/ I/A] (SEQ ID NO: 21) and Wl N -X4-GS (SEQ ID NO: 22), wherein X4 is any amino acid and a junction sequence between SEQ ID NO: 21 and SEQ ID NO: 22 of 4-7 amino acids.

It further relates to the use of a radical SAM peptide epimerase having an amino acid sequence that has at least 30% identity with SEQ ID NO: 25 and in which 70% of positions 14-15, 18 , 20-24, 27, 58-60, 63, 83, 84, 87-90, 112, 115, 117, 120, 150, 152, 169, 176, 180, 181, 183, 204, 206-208, 214 , 217, 220-225, 228, 230, 252, 254, 262, 289, 292, 296, and 309 of SEQ ID NO: 25 are conserved for the preparation of a peptide according to the present description.

The present disclosure also relates to a recombinant host cell comprising a heterologous nucleic acid encoding a peptide comprising a 17-residue sequence having at least 40% identity to the sequence at positions 33 to 49 of SEQ ID NO: No. 1 a and said sequence comprising the sequences WYF-[V/I/A] (SEQ ID No. 71) and W-[l N /A]-X4-GS (SEQ ID No. 69), in wherein least 30% identity with SEQ ID NO: 25 and in which 70% of positions 14-15, 18, 20-24, 27, 58-60, 63, 83, 84, 87-90, 112, 115, 117, 120, 150, 152, 169, 176, 180, 181, 183, 204, 206-208, 214, 217, 220-225, 228, 230, 252, 254, 262, 289, 292, 296 and 309 of SEQ ID NO: 25 and which is capable of co-expressing the peptide and the epimerase. In one aspect, the peptide comprises a 24-residue sequence having at least 40% identity with the sequence at positions 26 to 49 of SEQ ID NO: 1 and said sequence comprises the sequences N/DDLWYF-[V/ I/A] (SEQ ID NO: 21) and WI N -X4-GS (SEQ ID NO: 22), wherein X4 is any amino acid, and a junction sequence between SEQ ID NO: 21 and SEQ ID NO: 22, of 4 7 amino acids.

Finally, the present description relates to a method for preparing a peptide according to the present description, wherein the method comprises culturing a recombinant host cell according to the present description under conditions suitable for co-expression of the peptide and the epimerase. and recovering the peptide that has at least one amino acid with D configuration or synthetic methods used for the synthesis of peptides, which allow the assembly of amino acids with L and D configuration

DETAILED DESCRIPTION OF THE INVENTION

By studying the yydFGHIJ operon of Bacillus subtilis, the inventors identified firstly a new class of antimicrobial peptides and, secondly, an epimerase capable of converting in vitro an L-configuration amino acid into a D-configuration residue in the peptide. Therefore, this epimerase is useful for peptide engineering.

Antimicrobial peptide

In a search for genes involved in the regulation of the LiaRS two-component system presumably involved in sensing bacterial cell wall integrity, Butcher et al. (2007, J. Bacteriol., 189 , 8616) identified a YydFGHIJ operon that appeared to induce LiaRS expression. However, no component of this operon could be isolated or investigated in vitro.

The inventors showed that the operon produces a YydF peptide and that this peptide is post-translationally modified by the enzyme YydG which, very surprisingly, encodes a new class of radical SAM epimerase. Furthermore, the inventors discovered that the YydF peptide has antimicrobial activity but only if it comprises D-configuration amino acids. In the absence of D-configuration amino acids, the peptide is devoid of this antimicrobial activity, a characteristic unknown to date in bioactive peptides. More particularly, two D-configuration amino acids are required for this antimicrobial activity.

Accordingly, in one aspect, the present disclosure relates to a peptide having at least one D-configuration amino acid, preferably two D-configuration amino acids, comprising a sequence of 17 residues having at least 40% identity with the sequence at positions 33 to 49 of SEQ ID NO: 1. In particular, the peptide may comprise a sequence of 17 residues having at least a 45, 50, 55, 60, 70, 80, 85, 90, 95 , 98 or 99% identity with the sequence at positions 33 to 49 of SEQ ID NO: 1. Alternatively, the peptide may comprise a sequence of 17 residues at positions 33 to 49 of SEQ ID NO: 1, and 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, additions or deletions.

In a preferred aspect, the peptide comprises a sequence in which residues at positions 33, 35, 40-43, 46 and 47 of SEQ ID NO: 1 are conserved. In a further aspect, residues at positions 36 and 44 of SEQ ID NO: 1 are selected from the group consisting of V, I and A, preferably V and I.

In a preferred aspect, the present description relates to a peptide having at least two amino acids with a D configuration, comprising a sequence of 17 residues that has at least 40% identity with the sequence at positions 33 to 49 of the SEQ ID NO: 1 and including the sequence W-[l N /A]-X4-GS (SEQ ID NO: 69), wherein X4 is a hydrophobic amino acid, preferably leucine, valine or alanine. In particular, the peptide may comprise a sequence of 17 residues that have at least 40, 45, 50, 55, 60, 70, 80, 85, 90 or 95% identity with the sequence at positions 33 to 49 of the SEQ ID NO: 1. Optionally, the peptide further comprises the sequence of WYF-[V/I/A] (SEQ ID NO: 71). Preferably, the amino acids [V/I/A] of SEQ ID NOs 69 and 71 are D-configuration amino acids. In the peptide sequence, when considering the orientation from the N-terminal end to the C-terminal end, the sequence WYF-[V/I/A] (SEQ ID NO: 71) is before the sequence W-[l N /A]-X4-GS (SEQ ID NO: 69), preferably W-[l N ]-X4-GS (SEQ ID NO: 22). In a preferred aspect, the WYF-[V/I/A] motif (SEQ ID NO: 71) is separated from the W-[l N /A]-X4-GS motif (SEQ ID NO: 69), preferably W-[l N ]-X4-GS (SEQ ID NO: 22), by a binding sequence comprising four to eight amino acids, preferably a binding sequence of five or seven amino acids, preferably six amino acids. In a more preferred aspect, the peptide comprises a sequence of WYF-[V/I/A]-[K/R]-Xa-Xb-Xc-NRW-[V/I/A]-Xd-GS-Xe- H (SEQ ID NO: 72), wherein Xa, Xb and Xc are a polar amino acid, Xd is an aliphatic amino acid and Xe is any amino acid. Preferably, Xa is selected from the group consisting of S, N, C and T, more preferably it is S or N. Preferably, Xb is selected from the group consisting of S, N, T, Q, D, E, K, R and H, more preferably selected from the group consisting of S, N, Q and K, even more preferably S, Q and K. Preferably, Xc is selected from the group consisting of S, N, T, Q, D, E, K, R and H, more preferably selected from the group consisting of S, N, Q, E, R and K, even more preferably E, K, S and Q. Preferably, Xd is selected from the group consisting in L, I, VA, more preferably L, V and A. Preferably, Xe is selected from the group consisting of A, G or S, more preferably A or G.

In a very specific aspect, the peptide essentially consists of or consists of a sequence of WYF-[V/I/A]-[K/R]-[S/N]-[S/Q/K]-[E/K /S/Q]-NRW-[V/I/A]-[L N /A]-GS-[ N G]-H (SEQ ID NO: 73).

In one aspect of the description, the peptide comprises, essentially consists of or consists of a sequence selected from the group consisting of Ec ID Nos. 61-68.

In one aspect, the present disclosure relates to a peptide having at least one D-configuration amino acid, preferably two D-configuration amino acids, comprising a sequence of 24 residues having at least 40% identity with the sequence in the positions 26 to 49 of SEQ ID NO: 1. In particular, the peptide may comprise a sequence of 24 residues having at least 45, 50, 60, 70, 80, 85, 90, 95, 98 or 99% of identity with the sequence at positions 26 to 49 of SEQ ID NO: 1. Alternatively, the peptide may comprise a sequence of 24 residues at positions 26 to 49 of SEQ ID NO: 1, and 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, additions or deletions.

In a preferred aspect, the peptide comprises a sequence in which residues at positions 26, 31-35, 43, 46 and 47 of SEQ ID NO: 1 are conserved. In a further aspect, one or more residues in Positions 23, 25, 27-30, 36, 37, 38, 41, 42, 44, 48 and 49 of SEQ ID NO: 1 are also retained. For example, 1-11 residues at positions 23, 25, 27-30, 36, 37, 38, 41, 42, 44, 48 and 49 of SEQ ID NO: 1 can be retained, especially 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 remains. More preferably, the residues at positions 23, 25, 27-30, 36, 37, 38, 41, 42, 44, 48 and 49 of SEQ ID NO: 1 are retained. Even more preferably, the residues at positions 25, 29, and 38 of SEQ ID NO: 1 are conserved.

Next, a peptide according to the present description comprises the sequences N/DDLWYF-[V/I/A] (SEQ ID NO: 21), in particular L-X1-X2-X3-NDLWYFV/I (SEQ ID NO: 23), or/and W-IN/A-X4-GS (SEQ ID NO 22), wherein X1, X2, X3 and X4 are any amino acid. Optionally, the peptide comprises both sequences. Optionally, the sequence WI N /A-X4-GS (SEQ ID NO: 69) is the sequence of W-lN-X4-GS (SEQ ID NO: 22). Preferably, X1 is alanine, aspartic acid or glutamic acid, preferably alanine and aspartic acid, more preferably alanine. Preferably, X2 is lysine, asparagine or aspartic acid, preferably lysine or asparagine, more preferably lysine. Preferably, X3 is valine, isoleucine or glutamine, preferably valine or isoleucine, more preferably valine. Preferably, X4 is a hydrophobic amino acid, preferably leucine, valine, alanine or methionine. In a particular aspect, X1 is alanine and aspartic acid, preferably alanine, X2 is lysine or asparagine, preferably lysine, X3 is valine or isoleucine, preferably valine, and In a preferred aspect, the peptide comprises the sequences LAKVNDLWYFV (SEQ ID NO: 24), and W-IN-X4-GS (SEQ ID NO: 22), wherein X4 is a hydrophobic amino acid, preferably leucine, valine or alanine. “IN” or “V/l” means herein isoleucine or valine. “N/A” means herein asparagine or aspartic acid. “IN/A” means herein isoleucine, valine or alanine.

In a preferred aspect, in the sequence W-IN-X4-G-S (SEQ ID NO: 22), lN is an isoleucine (SEQ ID NO: 57). Alternatively, in the sequence W-IN-X4-G-S (SEQ ID NO:22), lN is a valine (SEQ ID NO:58). In another aspect, W-IN/A-X4-G-S (SEQ ID NO: 69) is W-A-X4-G-S (SEQ ID NO: 70). Preferably, X4 is a hydrophobic amino acid, more preferably leucine, valine, alanine or methionine, even more preferably leucine, valine or alanine.

In a preferred aspect, in the sequence N/D-D-L-W-Y-F-[V/I/A] (SEQ ID NO: 21) and N/D is asparagine and lN is valine. Alternatively, in the sequence of N/D-D-L-W-Y-F-[V/I/A] (SEQ ID NO: 21) and N/D is aspartic acid and IN is isoleucine.

In the peptide sequence, when considering the orientation from the N-terminal end to the C-terminal end, the sequence N/D-D-L-W-Y-F-[V/I/A] (SEQ ID NO: 21), in particular L-X1- X2-X3-N-D-L-W-Y-F-V/I (SEQ ID NO: 23), is before the sequence W-IN-X4-G-S (SEQ ID NO: 22).

In a preferred embodiment, the N/D-D-L-W-Y-F-[V/I/A] motif (SEQ ID NO: 21) is separated from the W-IN-X4-G-S motif (SEQ ID NO: 22) by a linker sequence. comprising four to seven amino acids, preferably, a binding sequence of five or six amino acids, preferably five amino acids. In the most preferred aspect, the peptide comprises a sequence of N/D-D-L-W-Y-F-V/I-(X)5-W-IN-X4-G-S (SEQ ID NO: 59) or N/D-D-L-W-Y-F-V/I-(x)6- W-IN-X4-G-S (SEQ ID NO. 60), X4 has the same meaning as before.

Therefore, in a preferred aspect, the present description relates to a peptide having at least one amino acid with D configuration, comprising a sequence of 24 residues that has at least 40% identity with the sequence at positions 26 to 49 of SEQ ID NO: 1 and includes the sequences LAKVNDLWYFV (SEQ ID NO: 24) and/or WI N -X4-GS (SEQ ID NO: 22), wherein X4 is a hydrophobic amino acid, preferably leucine, valine or alanine. In particular, the peptide may comprise a sequence of 24 residues having at least 45, 50, 60, 70, 80, 85, 90 or 95% identity with the sequence at positions 26 to 49 of SEQ ID NO: 1. In a preferred aspect, one or more residues at positions 23, 25, 27-30, 36, 37, 38, 41, 42, 44, 48 and 49 of SEQ ID NO: 1 are also conserved, especially 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 remains.

The peptide comprises at least one amino acid with D configuration, preferably two amino acids with D configuration. For example, it may comprise 1, 2, 3, 4 or 5 amino acids with D configuration. Optionally, the amino acids with D configuration can be any amino acid, preferably an amino acid selected from the group consisting of I, V, A, N, S and T, more preferably selected from the group consisting of I, V and A, even more preferably selected from the group consisting of I and V.

In a particular aspect, the amino acid valine, isoleucine or alanine in the sequence N/DDLWYF- [V/I/A] (SEQ ID NO: 21) has a D configuration and/or the isoleucine, valine or alanine in the sequence W-[lN/A]-X4-GS (SEQ ID NO: 69), preferably W- IN -X4-GS (SEQ ID NO: 22), has a D configuration. In a preferred aspect, the amino acids valine , isoleucine or alanine in the sequence N/DDLWYF- [V/I/A] (SEQ ID NO: 21) and isoleucine, valine or alanine in the sequence W-[lN/A]-X4-GS (SEQ ID No. 69), preferably W- IN -X4-GS (SEQ ID NO: 22), have a D configuration. In a preferred aspect, valine or isoleucine in the sequence of N / DDLWYF- V / l -( X) 5-W- L N-X4-GS (SEC ID No. 59) O N/DDLWYF- V/L- (X) 6-W- I N-X4-GS (SEC ID N. 60) They have a D configuration.

Optionally, the peptide may comprise an additional amino acid with D configuration.

Preferably, the length of the peptide is not more than about 65 amino acids, more preferably not more than 50 amino acids, in particular it may be about 15 to about 50 amino acids, for example, about 17 to about 50 amino acids, about 18 to about 50 amino acids, from about 17 to about 40, from about 20 to about 40 or from about 25 to about 35 amino acids.

The peptide may comprise, consist essentially of or consist of a sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 1-19 or a functional fragment thereof comprising the sequences N/DDLWYF-[ V/I/A] (SEQ ID NO: 21), in particular L-X1-X2-X3-NDLWYFV/I (SEQ ID NO: 23), and/or WI N -X4-GS (SEQ ID NO: 22), more preferably LAKVNDLWYFV (SEQ ID NO 24), and W-IN-X4-GS (SEQ ID NO 22). By functional we mean that it has antimicrobial activity, especially antibacterial activity.

In a very particular aspect, the peptide comprises, consists essentially of or consists of the sequence of SEQ ID NO: 20 and the valine at position 19 is in D configuration and/or the isoleucine at position 27 is in D configuration. Preferably, both valine at position 19 and isoleucine at position 27 are in D configuration.

In another very particular aspect, the peptide has the sequence of SEQ ID NO: 1 and the valine at position 36 is in D configuration and/or the isoleucine at position 44 is in D configuration. Preferably, both the valine in the position 36 and isoleucine at position 44 are in D configuration.

In a particular aspect, the peptide is not found in nature. The peptide is a non-natural peptide.

The N and C termini of the peptides described herein may optionally be protected from proteolysis. In a preferred aspect, the N-terminal end may be in the form of an acetyl group, and/or the C-terminal end may be in the form of an amide group. In a preferred aspect, the peptide has a free C-terminal end.

Alternatively or in addition, internal modifications of the peptides that are resistant to proteolysis are also included, for example in which at least one peptide bond -CONH- is modified and replaced by a reduced bond (CH ₂ NH), a retroinverse bond (NHCO), a methylene-oxy bond (CH ₂ -O), a thiomethylene bond (CH ₂ -S), a carba bond (CH ₂ CH ₂ ), a ketomethylene bond (CO-CH ₂ ), a hydroxyethylene bond ( CHOH-CH ₂ )), a (NN) bond, an E-alkene bond or also a -CH=CH- bond.

For example, the peptide can be modified by acetylation, acylation, amidation, cross-linking, cyclization, disulfide bond formation, covalent cross-link formation, cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, phosphorylation, and the like.

The peptide according to the description may comprise one or more amino acids that are rare amino acids, in particular hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, alloisoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid; or synthetic amino acids, in particular, ornithine, norleucine, norvaline and cycyclohexyl-alanine.

Optionally, the peptide may be linked to an additional moiety, optionally through a linker or spacer (e.g., diglycine). Optionally, the peptide may be part of a fusion protein. The additional residue may be a residue that facilitates its uptake or entry into the cell, in particular a PTD (protein transduction domain) or cell penetrating peptide; a localizing peptide; a stabilizing agent, such as PEG (polyethylene glycol), oligo-N-methoxy-ethylglycine (NMEG), albumin, an albumin binding protein or an immunoglobulin Fc domain; an affinity marker, such as an immune marker, biotin, lectin or chelator; a purification tag, such as a His tag; a detectable label, such as an optical label, a chelated lantamide, a fluorescent dye or a FRET acceptor/donor; a rest of addressing; a secretion signal peptide; or a combination thereof. The additional residue can be added either at the N-terminus or at the C-terminus of the peptide. Preferably, the additional residue is added either at the N-terminal end of the peptide.

In another aspect of the description, the peptides are covalently linked to a polyethylene glycol (PEG) molecule by its C-terminal end or a lysine residue, in particular a PEG of 1,500 or 4,000 MW, for a decrease in urinary clearance and in therapeutic doses used and for an increase in the half-life in blood plasma. In yet another aspect, the half-life of the peptide is increased by inclusion of the peptide in a biodegradable and biocompatible polymeric material for the microsphere-forming drug delivery system. The polymers and copolymers are, for example, poly(D,L-lactide-co-glycolide) (PLGA) (as illustrated in US2007/0184015, SoonKap Hahn et al.).

The description also encompasses pharmaceutically acceptable salts of a peptide according to the description. Pharmaceutically acceptable salts may, for example, be salts with pharmaceutically acceptable mineral acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid; salts of pharmaceutically acceptable organic acids, such as acetic acid, citric acid, maleic acid, malic acid, succinic acid, ascorbic acid and tartaric acid; salts with pharmaceutically acceptable mineral bases, such as sodium, potassium, calcium, magnesium or ammonium salts; or salts of organic bases containing a salifiable nitrogen, usually used in pharmaceutical technology. Methods for preparing said salts are well known to one skilled in the art.

In a preferred embodiment, the peptide is isolated.

Use of antimicrobial peptide

The present description relates to a pharmaceutical composition comprising a peptide as defined above. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and/or excipient. For pharmaceutical use, veterinary use is also contemplated. Optionally, the pharmaceutical composition may further comprise another active ingredient, preferably another antimicrobial agent, more preferably another antibacterial or an anti-inflammatory.

The pharmaceutical composition comprising the molecule is formulated in accordance with standard pharmaceutical practice (Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known to an expert in the technique.

The present description also relates to a peptide as defined above for use as a medicament, in particular as an antimicrobial agent, more preferably as an antibacterial. It further relates to the use of a peptide as defined above for the manufacture of a medicament for use as an antimicrobial agent, more preferably as an antibacterial. Furthermore, it relates to a method of treating or preventing an infection, especially a bacterial infection, comprising administering a therapeutically effective amount of a peptide as defined above, thereby treating or preventing an infection.

In a preferred aspect, the peptide as defined above is such that valine, isoleucine or alanine in the sequence N/DDLWYF- [V/I/A] (SEQ ID NO: 21) and isoleucine, valine or alanine in the sequence W-[lN/A]-X4-GS (SEQ ID NO: 69), preferably W- IN -X4-GS (SE C ID NO: 22), have a D configuration. In a very specific, the peptide comprises, consists essentially of or consists of the sequence of SEQ ID NO: 20 and the valine at position 19 and isoleucine at position 27 are in D configuration. In another very particular aspect, the peptide has the sequence of SEQ ID NO: 1 and valine at position 36 and isoleucine at position 44 are in D configuration. In another very particular aspect, the peptide comprises, essentially consists of or consists of a sequence selected from the group that consists of SEQ ID NO: 61-68, in which the residues at positions 4 and 12 are in D configuration.

Optionally, the peptide as defined above may be used in combination with another drug, preferably another antimicrobial, more preferably another antibacterial or an anti-inflammatory agent.

By “treat” or “treatment” it is understood that the disease is cured, alleviated or delayed. Includes preventive or curative treatment.

The term "therapeutically effective amount" as used in the present application means an amount of therapeutic agent, administered to a patient that is sufficient to constitute a treatment of the infection.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc.

The pharmaceutical compositions of the description can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.

For oral administration, the composition can be formulated in conventional oral dosage forms, such as tablets, capsules, powders, granules and liquid preparations, such as syrups, elixirs and concentrated drops. Non-toxic solid carriers or diluents may be used including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium, carbonate and the like. For tablets, binders are also necessary, which are agents that impart cohesive qualities to powder materials. For example, starch, gelatin, sugars, such as lactose or dextrose, and natural or synthetic gums can be used as binders. Disintegrants are also necessary in tablets to facilitate tablet disintegration. Disintegrants include starches, clays, celluloses, algins, gums and cross-linked polymers. In addition, lubricants and glidants are also included in the tablets to prevent adhesion of the tablet material to surfaces in the manufacturing process and to improve the flow characteristics of the powder material during manufacturing. Colloidal silicon dioxide is most commonly used as a glidant and compounds such as talc or stearic acid are most commonly used. as lubricants.

For transdermal administration, the composition can be formulated in the form of an ointment, cream or gel and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide.

For transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used. The active compound can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carboceras), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate.

Pharmaceutical compositions according to the disclosure may be formulated to release the active drug substantially immediately upon administration or at any predetermined time or period of time upon administration.

In a particular aspect, the pharmaceutical composition according to the description comprises from 0.001 mg to 10 g of the molecule of the description. Preferably, the pharmaceutical composition according to the description comprises 0.01 mg to 1 g of the molecule of the description.

In yet another aspect, the present description relates to the use of a peptide according to the description as a disinfectant, preservative or pesticide. The term “disinfectant” refers to an antimicrobial activity of the peptide on a surface (e.g., walls, doors, medical equipment), a liquid (e.g., water), or a gas (e.g., an anesthetic gas). According to one aspect, the peptide according to the description is used for the removal of bacterial biofilms. According to a preferred aspect, the peptide according to the description is used in particular for the disinfection of surgical or prosthetic equipment.

In another aspect, the present description relates to a medical device or implant comprising a body having at least one surface coated with or including a peptide according to the description. The present description also relates to a method of preparing a medical device or implant comprising applying a peptide coating according to the description, or contacting, with at least one surface of the device or implant.

This type of medical device or implant and the uses and preparation methods thereof are described, for example, in patent application WO 2005/006938.

The surface coated with or including a peptide according to the description may be composed of thermoplastic or polymeric materials such as polyethylene, dacron, nylon, polyesters, polytetrafluoroethylene, polyurethane, latex, silicone elastomers and the like, or of metallic materials such as gold. In a particular aspect, the peptide of the invention is covalently linked to a functionalized surface, preferably a metallic surface, through its N-terminal or C-terminal end. Optionally, the peptide may be attached to the surface via a spacer arm.

Preferably, the surface may be coated with a peptide at a density of 0.4 to 300 mg/cm2.

Alternatively, the device or implant, in particular the bone and joint prosthetic device, may be coated with a cement mixture comprising a peptide according to the description.

The peptide can be combined with another active molecule, preferably an antibiotic.

The device or implant can be, for example, intravascular, peritoneal, pleural and urological catheters; Heart valves; pacemaker; vascular bypasses; coronary bypasses; dental implants or orthopedic or intraocular prostheses.

Peptide preparation

The peptides described herein can be synthesized using standard synthetic methods known to those skilled in the art, for example chemical synthesis or genetic recombination. In particular, peptides can be synthesized according to the method originally described by Merrifield.

Examples of chemical synthesis technologies are solid phase synthesis and liquid phase synthesis. As a solid phase synthesis, for example, the amino acid corresponding to the C-terminal end of the peptide to be synthesized is attached to a support that is insoluble in organic solvents, and by alternating repetition of reactions, one in which the amino acids with its amino groups and side chain functional groups protected with appropriate protecting groups are condensed one by one in order from the C-terminus to the N-terminus, and one where the amino acids attached to the resin or the protecting group of the amino groups of The peptides are released, thus, in this way the peptide chain extends. Solid phase synthesis methods are mainly classified by the tBoc method and the Fmoc method, depending on the type of protecting group used. Commonly used protecting groups include tBoc (t-butoxycarbonyl), Cl-Z (2-chlorobenzyloxycarbonyl), Br-Z (2-bromobenzyloxycarbonyl), Bzl (benzyl), Fmoc (9-fluorenylmethoxycarbonyl), Mbh (4,4'-dimethoxydibenzhydryl), Mtr (4-methoxy-2,3,6-trimethylbenzenesulfonyl), Trt (trityl), Tos (tosyl), Z (benzyloxycarbonyl) and Clz-Bzl (2,6-dichlorobenzyl) for the amino groups; NO ₂ (nitro) and Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl) for guanidino groups); and tBu (t-butyl) for hydroxyl groups). After synthesis of the desired peptide, it is subjected to the deprotection reaction and cut from the solid support. Such peptide cleavage reaction can be carried out with hydrogen fluoride or trifluoromethanesulfonic acid for the Boc method and with TFA for the Fmoc method.

Alternatively, the peptide can be synthesized using recombinant techniques. In this case, a nucleic acid and/or a genetic construct is used comprising a nucleotide sequence encoding a peptide according to the description. Therefore, the present description relates to a nucleic acid and/or a genetic construct comprising a nucleotide sequence encoding a peptide according to the description. The genetic construct comprises a polynucleotide encoding a peptide according to the description as defined herein and regulatory sequences (such as one or more suitable promoters, enhancers, terminators, etc.), allowing expression (e.g. example, transcription and translation) of a peptide according to the description in a host cell. Thus, in another aspect, the invention relates to a host or host cell that expresses (or, in suitable circumstances, is capable of expressing) a peptide of the invention; and/or containing a polynucleotide of the description or genetic construct of the description.

In order to obtain the D-amino acid containing peptide, the peptide can be co-expressed with the peptide epimerase described herein.

The method for producing the peptide may optionally comprise the steps of purification of said peptide, chemical modification of said peptide and/or formulation of said peptide in a pharmaceutical composition. For example, the peptide described herein can be prepared by recombinant techniques as a fusion protein with a secretion signal peptide or a purification marker. This secretion signal peptide or purification marker can be cleaved or removed in a further production step.

In a particular object of the present description, the method of preparing the peptide comprises providing or synthesizing a peptide as described above with the amino acids in L configuration and contacting the peptide with an enzyme (i.e., an epimerase), converting thus at least one amino acid in L configuration in amino acid in D configuration of said peptide, the epimerase is as defined below.

radical SAM epimerase

The inventors have identified a radical SAM epimerase called YydG in Bacillus subtilis that is capable of modifying the configuration of the amino acids contained in a peptide from an L configuration to a D configuration. This enzyme is capable of carrying out the conversion, even under conditions in vitro. Its sequence is shown in SEQ ID NO: 18. Furthermore, this enzyme is not related to other known epimerases, even within the radical SAM enzyme superfamily.

Therefore, the present description refers to an epimerase capable of modifying the configuration of the amino acids contained in a peptide from an L configuration to a D configuration and comprising an amino sequence that has at least 30% identity with the SEC ID NO: 25. Preferably, the epimerase comprises an amino sequence that has at least 35, 40, 50, 60, 70, 80, 85, 90, 95, 97.5 or 99% identity with SEQ ID #25.

YydG from Bacillus subtilis is described in public databases with the following identification numbers: UniProt Q45595; GenelD 937720; GenBank NP_391897.1 and NC_000964.3.

According to the teachings of the present disclosure, the person skilled in the art can identify other enzymes from microorganisms that have the radical SAM epimerase. The polypeptide can be identified and obtained from other sources, including microorganisms isolated from nature (for example, soil, fertilizers, water, etc.) or DNA samples obtained directly from natural materials (for example, soil, fertilizers, water , etc.) using the probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. Therefore, a polynucleotide encoding the polypeptide can be obtained by similarly screening a genomic DNA or cDNA library from another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected, the polynucleotide can be isolated or cloned using techniques that are known to those skilled in the art (see, for example, Sambrook et al., 1989). The person skilled in the art could also use data sequences already available in databases. Furthermore, one skilled in the art can prepare epimerase variants having the amino acid sequence of SEQ ID NO:25 by currently used methods. In particular, variants with advantageous properties such as increased stability (e.g., thermostability), increased D-configuration amino acid production, modification of the specificity and/or selectivity of the epimerase, and the like.

100% identical sequences compared to SEQ ID NO: 25 are disclosed in Uniprot AOAOC ₂ UHS5, AOAOA1MJP5 and L8AWU7. Other strains of Bacillus subtilis have sequences with high identity (up to 90% identity) and are described in SEQ ID NOs 26-29 and 32. Other strains of Bacillus have sequences with high identity and are disclosed in SEQ ID NO: 26-29 and 32. ID Nos. 30 and 33-35. Sequences with significant identity have been identified in Salinibacillus aidiingensis, Staphylococcus equorum, Staphylococcus pseudintermedius, Staphylococcus epidermidis, Paenibacillus sp, Enterococcus caccae, Enterococcus faecalis, Corynebacterium diphtheria, Streptococcus agalactiae and Bifidobacterium bohemicum and are disclosed in SEQ ID NOs 31, and 36-52. Figure 6 shows an alignment of the enzyme sequences.

The positions in bold and underlined are perfectly preserved among the list of sequences revealed above. The positions in bold and italics are conserved among groups of strongly similar properties in the list of sequences disclosed above. Therefore, it can be seen that even if the percentage of identity is approximately 30%, there are a high number of amino acids that are perfectly conserved and well conserved.

Accordingly, in a preferred embodiment, the epimerase has an amino acid sequence that has at least 30% identity with SEQ ID NO: 25 and in which 70, 80 or 90% of the bold and underlined positions are conserved when the sequence is aligned to SEQ ID NO: 25 (positions 14-15, 18, 20-24, 27, 58-60, 63, 83, 84, 87-90, 112, 115, 117, 120 , 150, 152, 169, 176, 180, 181, 183, 204, 206-208, 214, 217, 220-225, 228, 230, 252, 254, 262, 289, 292, 296, and 309 of the SEC ID #25). Preferably, 95% of the bold and underlined positions are preserved. In a particular aspect, all bold and underlined positions are preserved. More preferably, 90 or 95% of positions 14-15, 18, 20-24, 27, 58-60, 63, 83, 84, 87-90, 112, 115, 117, 120, 204, 206-208 , 214, 217, 220-225, 228 and 230 of SEQ ID NO: 25 are conserved in the epimerase sequence. In a particular aspect, all positions 14-15, 18, 20-24, 27, 58-60, 63, 83, 84, 87-90, 112, 115, 117, 120, 204, 206-208, 214, 217, 220-225, 228 and 230 of SEQ ID NO: 25 are conserved in the epimerase sequence.

In a preferred aspect, the epimerase has an amino acid sequence that has at least 30% identity with SEQ ID NO: 25 and one or more segments selected from segments 13-27, 56-63, 83-90, 112-120, and 204,230 have at least 50% identity with the sequence of SEQ ID NO: 25. In this aspect, the epimerase also has 90 or 95% of positions 14-15, 18, 20 -24, 27, 58-60, 63, 83, 84, 87-90, 112, 115, 117, 120, 204, 206 208, 214, 217, 220-225, 228 and 230 of SEQ ID NO: 25 are conserved in the epimerase sequence.

In a particular aspect, cysteine residues at positions 14, 18, 21, 222 and 223 of SEQ ID NO: 25 are conserved in the epimerase sequence.

In one aspect, the epimerase has an amino acid sequence comprising, essentially consisting of, or consisting of, a sequence that has at least 80, 85, 90, or 95% identity with a sequence selected from SEQ ID NOs. 25-52, preferably between SEQ ID NO: 25-30, 32 and 34, more preferably SEQ ID NO: 25. The epimerase may have an amino acid sequence comprising, consisting essentially of, or consisting of , a sequence selected from SEQ ID NO: 25-52, preferably from SEQ ID NO: 25-30, 32 and 34, more preferably SEQ ID NO: 25; and that has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, additions or deletions.

A method for analyzing the ability of an epimerase to modify the configuration of the amino acids contained in a peptide from an L configuration to a D configuration is disclosed, for example, in the details in the examples section. For example, the epimerase is contacted with a peptide having a sequence selected from the group consisting of SEQ ID NOs: 1-20 in the presence of the S-adenosyl-L-methionine (SAM) cofactor and the production of peptides, including a D-configuration amino acid. More specifically, the epimerase is contacted with the peptide YydF18-49 of SEQ ID NO: 20 in the presence of the S-adenosyl-L-methionine (SAM) cofactor and is detects the production of peptides that include a D-configuration amino acid at position 19 and/or 27.

Also provided is a hybrid polypeptide or fusion polypeptide in which the amino acid sequence of the enzyme as defined above is fused to the N-terminus or the C-terminus of a region of another polypeptide. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences that encode the enzyme and the addition region of another polypeptide so that they are in frame and that the expression of the fusion polypeptide is under the control of the same promoter or promoters and terminator. Fusion polypeptides can also be constructed using intein technology, in which fusion polypeptides are created after translation (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

The addition region of the fusion polypeptide can be selected in order to improve the stability of the enzyme according to the present disclosure, to stimulate the secretion (such as an N-terminal hydrophobic signal peptide) of the fusion protein. a cell (such as a bacterial cell or a yeast cell), or to assist in the purification of the fusion protein. More particularly, the additional region may be a useful marker for purification or immobilization of the enzyme. Such a marker is well known to the person skilled in the art, for example, a His tag (His6), a FLAG tag, an HA tag (epitope derived from Influenza hemagglutinin protein), a maltose binding protein (MPB), a MYC marker (epitope derived from the human MYC proto-oncoprotein), a STREP marker or a GST (small glutathione-S-transferase) marker.

A fusion polypeptide may further comprise a cleavage site between the enzyme and the addition region. Upon secretion of the fusion protein, the site cleaves and releases the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbio!. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbe!. 63:3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Further provided is a recombinant nucleic acid construct or vector comprising a nucleic acid sequence encoding epimerase as defined above. More particularly, the nucleic acid or vector construct is suitable for expressing said epimerase. Additionally, there is provided a recombinant host cell comprising a nucleic acid, a recombinant nucleic acid construct, or a recombinant vector comprising a nucleic acid sequence encoding epimerase as defined above.

Nucleic acid constructs

In fact, the present description relates to a polynucleotide encoding an epimerase of the present description. The nucleic acid can be DNA (cDNA or gDNA), RNA, or a mixture of the two. It can be in the form of a single chain or in the form of a duplex or a mixture of the two. It may comprise modified nucleotides, comprising, for example, a modified linker, a modified purine or pyrimidine base, or a modified sugar. It can be prepared by any method known to one skilled in the art, including chemical synthesis, recombination and mutagenesis.

The present description also relates to nucleic acid constructs comprising a polynucleotide encoding an epimerase according to the present description operably linked to one or more control sequences that direct expression of the coding sequence in a suitable host cell under conditions compatible with control sequences. A polynucleotide can be manipulated in various ways to provide expression of the epimerase. Manipulation of the polynucleotide prior to insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

The control sequence may include a promoter that is recognized by a host cell or an in vitro expression system for the expression of a polynucleotide encoding an epimerase of the present disclosure. The promoter contains transcription control sequences that participate in the expression of the epimerase. The promoter can be any polynucleotide that shows transcriptional activity in the host cell, including mutant, truncated and hybrid promoters, and can be obtained from genes that encode extracellular or intracellular polypeptides, either homologous or heterologous to the host cell. Optionally, the promoter may be inducible.

Examples of suitable promoters in a bacterial host cell are promoters obtained from the alpha-amylase gene of Bacillus amyloliquefaciens (amyQ), the alpha-amylase gene of Bacillus licheniformis (amyL), the penicillinase gene of Bacillus licheniformis (penP), the maltogenic amylase gene of Bacillus stearothermophilus (amyM), the levansucrase gene of Bacillus subtilis (sacB), the xylA and xylB genes of Bacillus subtilis, the crylllA gene of Bacillus thuringiensis (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), the lac operon of E. coli, the trc promoter of E. coli (Egon et al., 1988, Gene 69: 301-315), the agarase gene of Streptomyces coelicolor (dagA) and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Other promoters are described in “Useful proteins from recombinant bacteria” in Gilbert et al., 1980, Scientific American 242, 74-94; and in Sambrook et al., 1989. Examples of tandem promoters are disclosed in WO 99/43835.

Examples of suitable promoters in a filamentous fungal host cell are promoters obtained from the genes for acetamidase from Aspergillus nidulans, neutral alpha-amylase from Aspergillus niger, acid-stable alpha-amylase from Aspergillus niger, glucoamylase from Aspergillus niger or Aspergillus awamori ( glaA), TAKA amylase from Aspergillus oryzae, alkaline protease from Aspergillus oryzae, triose phosphate isomerase from Aspergillus oryzae, trypsin-like protease from Fusarium oxysporum (WO96/00787), amylglucosidase from Fusarium venenatum (Wo 00/56900), Daria from Fusarium venenatum (WO 00/56900), Quinn from Fusarium venenatum (WO 00/56900), lipase from Rhizomucor miehei, aspartic proteinase from Rhizomucor miehei, beta-glucosidase from Trichoderma reesei, cellobiohydrolase I from Trichoderma reesei, cellobiohydrolase II from Trichoderma reesei, endoglucanase I of Trichoderma reesei, endoglucanase II of Trichoderma reesei, endoglucanase III of Trichoderma reesei, endoglucanase IV of Trichoderma reesei, endoglucanase V of Trichoderma reesei, xylanase I of Trichoderma reesei, xylanase II of Trichoderma reesei, beta-xylosidase of Trichoderma reesei, as well as the NA2-tpi promoter (a promoter modified from an Aspergillus neutral alpha-amylase gene in which the untranslated leader sequence has been replaced by an untranslated leader sequence from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters of the neutral alpha-amylase gene of Aspergillus niger, in which the untranslated leader sequence has been replaced by an untranslated leader sequence of a triose phosphate isomerase gene of Aspergillus nidulans or Aspergillus oryzae; and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH ₂ /GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other promoters useful for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell can be used in the present description.

Preferred terminators for bacterial host cells are derived from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

Preferred terminators for host cells of filamentous fungi are obtained from the genes for anthranilate synthase of Aspergillus nidulans, glucoamylase of Aspergillus niger, alpha-glucosidase of Aspergillus niger, TAKA amylase of Aspergillus oryzae and trypsin-like protease of Fusarium oxysporum.

Preferred terminators for yeast host cells are derived from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, cited above.

The control sequence may also be an mRNA stabilizing region downstream of a promoter and upstream of the coding sequence of a gene that increases expression of the gene.

Examples of suitable mRNA stabilizing regions are obtained from a crylllA gene from Bacillus thuringiensis (WO 94/25612) and an SP82 gene from Bacillus subtilis (Hue et al. , 1995, Journal of Bacteriology 177: 3465-3471 ).

The control sequence can also be a leader, an untranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5' end of the polynucleotide encoding the epimerase. Any leader that is functional in the host cell can be used.

Preferential leaders for host cells of filamentous fungi are obtained from the TAKA amylase genes of Aspergillus oryzae and triose phosphate isomerase of Aspergillus nidulans.

Suitable leaders for yeast host cells are derived from the Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes. Saccharomyces cerevisiae (ADH ₂ /GAP).

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' end of the polynucleotide that encodes the epimerase and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell can be used.

The preferred polyadenylation sequences for host cells of filamentous fungi are obtained from the genes for anthranilate synthase of Aspergillus nidulans, glucoamylase of Aspergillus niger, alpha-glucosidase of Aspergillus niger, TAKA amylase of Aspergillus oryzae and trypsin-like protease of Fusarium oxysporum.

Polyadenylation sequences useful for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol 15:5983-5990.

The control sequence may also be a signal peptide coding region that encodes a signal peptide attached to the N terminus of the epimerase and directs the epimerase into the secretory pathway of the cell. The 5' end of the polynucleotide coding sequence may inherently contain a signal peptide coding sequence naturally linked in translational reading frame to the segment of the coding sequence that encodes the epimerase. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required when the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance secretion of the polypeptide. However, any signal peptide coding sequence can be used that directs the expressed polypeptide to the secretory pathway of a host cell.

The signal peptide coding sequences effective for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 of maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase , Bacillus licheniformis alpha-amylase. Bacillus stearothermophilus, neutral proteases from Bacillus stearothermophilus (nprT, nprS, nprM) and prsA from Bacillus subtilis. Other signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

The signal peptide coding sequences effective for host cells of filamentous fungi are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulose, endoglucanase V from Humicola insolens, lipase from Humicola lanuginosa and aspartic proteinase from Rhizomucor miehei.

Signal peptides useful for yeast host cells are derived from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, cited above.

It may also be desirable to add regulatory sequences that regulate expression of the polypeptide in relation to host cell growth. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH ₂ system or the GAL1 system can be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter, and the Aspergillus oryzae glucoamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate and the metallothionein genes that are amplified by heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked to the regulatory sequence.

expression vectors

The present disclosure also relates to recombinant expression vectors comprising a nucleic acid construct as disclosed above, or a polynucleotide encoding an epimerase of the present disclosure, a promoter, and transcription and translation termination signals. The various nucleotide and control sequences may be linked together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow insertion or substitution of the polynucleotide encoding the epimerase at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a vector appropriate for expression.

In a particular aspect, the expression vector may further comprise a polynucleotide encoding a peptide of the present invention as described above, operably linked to the control sequences necessary for its expression. More particularly, the control sequences used for expression of the epimerase and the peptide of the present disclosure are suitable for co-expression in a host cell. Optionally, the polynucleotide encoding the peptide and the polynucleotide encoding the epimerase may be under the control of a single promoter (i.e., operon) or two promoters that may be the same or different.

Alternatively, the present description relates to a kit comprising a first expression vector comprising a nucleic acid encoding an epimerase of the present description and a second expression vector comprising a nucleic acid encoding a peptide of the present description. . In another alternative, the kit may comprise an expression vector comprising a nucleic acid encoding an epimerase of the present description and a nucleic acid encoding a peptide of the present description.

In creating the expression vector, the coding sequence is located in the vector such that the coding sequence is operatively linked to the control sequences appropriate for expression.

The recombinant expression vector can be any vector (for example, a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will usually depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, for example, a plasmid, an extrachromosomal element, a mini-chromosome or an artificial chromosome. The vector may contain any means to ensure self-replication. Alternatively, the vector may be one that, when introduced into the host cell, integrates into the genome and replicates along with the chromosome(s) into which it has integrated. Additionally, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell or a transposon can be used.

The vector preferably contains one or more selectable markers that allow easy selection of transformed, transfected, transduced or similar cells. A selectable marker is a gene whose product provides biocidal or viral resistance, resistance to heavy metals, prototrophy to auxotrophy, and the like.

Examples of bacterial selectable markers are genes from Bacillus licheniformis or Bacillus subtilis or markers that confer resistance to antibiotics such as resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin or tetracycline. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Markers selectable for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase) and trpC (anthranilate synthase), as well as equivalents thereof. Preferably used in an Aspergillus cell are the amdS and pyrG genes from Aspergillus nidulans or Aspergillus oryzae and a gene from Streptomyces hygroscopicus.

The vector preferably contains an element or elements that allow integration of the vector into the genome of the host cell or autonomous replication of the vector in the cell independent of the genome.

When integration occurs into the genome of the host cell, the integration of the sequences into the genome can be based on homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct integration by homologous recombination into the host cell genome at one or more precise locations on the chromosome(s). To increase the probability of integration at a precise location, the integration elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs. that have a high degree of sequence identity with the corresponding target sequence to improve the probability of homologous recombination. Integrational elements can be any sequence that is homologous to the target sequence in the host cell genome. Additionally, the integrational elements may be non-coding or coding polynucleotides. On the other hand, the vector can integrate into the host cell genome by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicator that mediates autonomous replication functioning in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that allows a plasmid or vector to replicate in vivo . Examples of bacterial origins of replication are the replication origins of plasmids pBR322, pUC19, pACYC177, and pACYC184 that allow replication in E. coli, and pUB1 10, pE194, pTA1060, and pAMpI that allow replication in Bacillus. Examples of replication origins for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. Examples of useful replication origins in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be achieved according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present disclosure can be inserted into a host cell to increase production of a polypeptide. An increase in the number of copies of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or by including an amplifiable selectable marker gene with the polynucleotide where cells containing copies of the selectable marker gene amplify, and Thus, additional copies of the polynucleotide can be selected for culturing the cells in the presence of the appropriate selectable agent.

The procedures used to link the elements described above to construct the recombinant expression vectors of the present description are well known to one skilled in the art (see, for example, Sambrook et al., 1989, supra).

Host cells

The present description also relates to recombinant host cells comprising a polynucleotide encoding an epimerase according to the present disclosure operatively linked to one or more control sequences that direct production of the epimerase of the present description. A construct or vector comprising a polynucleotide encoding an epimerase according to the present disclosure is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as described above.

The present description further relates to a recombinant host cell further comprising a polynucleotide encoding a peptide according to the present disclosure operatively linked to one or more control sequences that direct production of the peptide. In particular, the host cell may coexpress the epimerase and the peptide according to the present disclosure. In this aspect, the host cell produces both a peptide of the present disclosure and epimerase that is capable of modifying the antimicrobial peptide by changing the amino acid configuration of the peptide from an L configuration to a D configuration.

In a preferred aspect, the host cell comprises a nucleic acid encoding an epimerase of the present invention heterologous to the host cell. In an alternative preferred aspect, the host cell comprises a nucleic acid encoding a peptide heterologous to the host cell. In another preferred aspect, the host cell comprises nucleic acids encoding a peptide and an epimerase, both heterologous to the host cell.

The term "host cell" encompasses any progeny of a parental cell that is not identical to the parental cell due to mutations that occur during replication. The choice of a host cell will largely depend on the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of an epimerase of the present disclosure, for example, a prokaryote or a eukaryote.

The prokaryotic host cell can be any gram-positive or gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus and Streptomyces. Grannegative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella and Ureaplasma. The bacterial host cell may be any Bacillus cell, including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis. The bacterial host cell may be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, Streptococcus equi and Streptococcus zooepidemicus . The bacterial host cell may further be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus and Streptomyces lividans.

The introduction of DNA into a Bacillus cell can be carried out by protoplast transformation (see, for example, Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see, for example, Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation (see, for example, Shigekawa and Dower, 1988 , Biotechniques 6: 742-751), or conjugation (see, for example, Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell can be carried out by protoplast transformation (see, for example, Hanahan, 1983, J. Mol. Biol.

166: 557-580), or electroporation (see, for example, Dower et al, 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell can be carried out by protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, for example, Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or transduction (see, for example, Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98, 6289-6294). The introduction of DNA into a Pseudomonas cell can be carried out by electroporation (see, for example, Choi et al 2006 J. Microbiol. Methods 64: 391-397) conjugation (see, for example, Pinedo and Smets, 2005, Appl. Environ. Microbio!. 71: 51-57). The introduction of DNA into a Streptococcus cell can be effected by natural competition (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, for example, Catt and Jollick, 1991, Microbios 68: 189-207), electroporation (see, for example, Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see, for example, Clewell, 1981, Microbiol. Rev. 45, 409-436). However, any method known in the art can be used to introduce DNA into a host cell.

The host cell may also be a eukaryotic cell, such as a mammalian, insect, plant or fungal cell. The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, as well as Oomycota and all mitosporic fungi (as defined by Hawksworth et al., in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition , 1995, CAB International, University Press, Cambridge, United Kingdom). The fungal host cell may be a yeast cell. “Yeast,” as used herein, includes ascosporogenic yeast (Endomycetales), basidiosporogenic yeast, and yeast belonging to Fungi imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this description, yeast will be defined as described in “Biology and Activities of Yeast” (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980). The yeast host cell may be a cell of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia , such as a cell of Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces ccharomyces norbensis, Saccharomyces oviformis or Yarrowia lipolytica . The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, cited above). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. The filamentous fungal host cell may be a cell of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete , Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes or Trichodermal. For example, the filamentous fungal host cell may be a cell of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium gueenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hir sutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum , Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola la nuginosa, Mucor Miehei, Myceliophthora Thermophila, Neurospora Crassa, Penicillium Purpurogenum, Phanerochaete Chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Villosa tramets, Versicolor tramets, trichoderma hazianum, trichoderma koningii, trichoderma longibrachiatum , Trichoderma Reesei or Viride Trichoderma.

Fungal cells can be transformed by a process involving protoplast formation, protoplast transformation and cell wall regeneration in a manner known per se. Suitable procedures for the transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Academic Sci. USA 81: 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described in Malardier et al, 1989, Gene 78: 147-156 and WO 96/00787. Yeast can be transformed using the procedures described by Becker and Guarente, in Abelson, JN and Simon, MI, editors, “Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology”, Volume 194, p. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Academic Sci. USA 75:1920.

The cell may also be a mammalian cell, for example, COS, CHO (US 4,889,803; US 5,047,335). In a particular aspect, the cell is non-human and non-embryonic. Furthermore, a non-human transgenic animal could produce the epimerase of the disclosure, for example in the milk produced by the animal.

The cell may be a plant cell. Therefore, the epimerase of the description could be the product of a transgenic plant.

Alternatively, there is also provided a method for producing an epimerase according to the present description, comprising culturing the host cell as defined above, under conditions conducive to the production of the epimerase and recovering and/or purifying the epimerase. Alternatively, a method for producing an epimerase according to the present description is also provided, comprising in vitro expression of the epimerase with a nucleic acid encoding the epimerase as defined above. Optionally, the method further comprises a step of immobilizing the epimerase on a solid support.

The enzyme can be recovered using methods known in the art. For example, the enzyme can be recovered from the nutrient medium by conventional procedures, including, but not limited to, harvesting, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.

The enzyme can be purified by various procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing and size exclusion), electrophoretic procedures (e.g. preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides . In an alternative aspect, the enzyme is not recovered, but rather a host cell of the present disclosure that expresses the enzyme is used as a source of the enzyme.

Also provided is a composition or a kit comprising the isolated or recombinant epimerase as defined above. In particular, the composition or kit may include iron (e.g., (NH4) _2Fe ( _SO4 ) ₂ ) and sulfur (e.g., ^Na2S ) as enzyme additives and a reducing agent, such as dithiothreitol or beta-mercaptoethanol. . Additionally, the composition may include S-adenosyl L-methionine (SAM), the cofactor of the enzyme, or methionine and ATP in the presence of SAM synthase. Optionally, the epimerase can be immobilized on a solid support.

The present description also relates to the use of an epimerase as defined above, a composition, a kit or solid support comprising the epimerase, or a recombinant host cell comprising a nucleic acid, a recombinant nucleic acid construct or a vector recombinant comprising a nucleic acid sequence encoding the epimerase as defined above, to produce the peptide with D configuration amino acids.

Using epimerase to convert L-amino acid to D-amino acid in a peptide

Accordingly, the present disclosure relates to the use of an epimerase of the disclosure to convert an L-amino acid to a D-amino acid in a peptide. It also relates to a method for converting L-amino acid to D-amino acid in a peptide comprising contacting an epimerase of the present description with the peptide and, optionally, recovering the peptide with at least one amino acid converted from an L configuration to a D configuration.

In a preferred aspect, the present description relates to a method for preparing a peptide according to the present description, wherein the method comprises a step of contacting a peptide according to the present description with an epimerase as set forth defined above. It also refers to the use of an epimerase as defined above to prepare a peptide according to the present description. In particular, the peptide may have any particular sequence as defined above. Preferably, the method is carried out in vitro. In a first aspect, the peptide comprises a 17-residue sequence that has at least 40% identity with the sequence at positions 33 to 49 of SEQ ID NO: 1 and said sequence comprises the sequences WYF-[V/ I/A] (SEQ ID NO: 71) and W-[l N /A]-X4-GS (SEQ ID NO: 69), wherein X4 is any amino acid, and a linker sequence between SEQ ID NO: ID NO: 69 and SEQ ID NO: 71 of 4-8 amino acids. In a second aspect, the peptide comprises a sequence of 24 residues having at least 40% identity with the sequence at positions 26 to 49 of SEQ ID NO: 1, and comprises the sequences of N/DDLWYF-[ V/I/A] (SEQ ID NO: 21) and WI N -X4-GS (SEQ ID NO: 22), where X4 is any amino acid.

Finally, the present description relates to a method for preparing a peptide according to the present description, wherein the method comprises culturing a recombinant host cell according to the present description under conditions suitable for co-expression of the peptide and the epimerase. and recovering the peptide having at least one amino acid with D configuration. Preferably, the method is carried out ex vivo. It also relates to the use of a recombinant host cell according to the present description for the preparation of a peptide as defined above.

In a particular aspect, the sources of epimerase and peptide may coincide. For example, an epimerase from Bacillus subtilis could preferably be used to prepare a peptide from Bacillus subtilis or derivative thereof. In this particular aspect, if the peptide as defined above comprises a sequence that has at least 80, 85, 90 or 95% identity with a sequence selected from SEQ ID NO: 1-5 or a fragment of the same of at least 24 consecutive residues, the chosen epimerase will have at least 80, 85, 90 or 95% identity with a sequence selected from SEQ ID NOs 25-35. If the peptide as defined above comprises a sequence that has at least 80, 85, 90 or 95% identity with a sequence selected from SEQ ID NO: 6-9 or a fragment thereof of at least 24 consecutive residues, the chosen epimerase will have at least 80, 85, 90 or 95% identity with a sequence selected from SEQ ID NOS 36-38. If the peptide as defined above comprises a sequence that has at least 80, 85, 90 or 95% identity with a sequence of SEQ ID NO: 10 or a fragment thereof of at least 24 consecutive residues, the epimerase chosen will have at least 80, 85, 90 or 95% identity with a sequence selected from SEQ ID NO: 39 40. If the peptide as defined above comprises a sequence that has at least 80, 85 , 90 or 95% identity with a sequence selected from SEQ ID NO: 11-13 and 17-18 or a fragment thereof of at least 24 consecutive residues, the chosen epimerase will have at least 80, 85, 90 o 95% identity with a sequence selected from SEQ ID NOS 41-50. If the peptide as defined above comprises a sequence that has at least 80, 85, 90 or 95% identity with a sequence selected from SEQ ID NO: 14-16 or a fragment thereof of at least 24 consecutive residues, the epimerase chosen will have at least 80, 85, 90 or 95% identity with a sequence of SEQ ID NO: 51. If the peptide as defined above comprises a sequence that has at least 80, 85, 90 or 95% identity with a sequence of SEQ ID NO: 19 or a fragment thereof of at least 24 consecutive residues, the epimerase chosen will have at least 80, 85, 90 or 95% identity with a sequence of SEQ ID NO:52.

Definitions

Approximately: When used herein, “approximately” means plus or minus 10%, preferably plus or minus 5%. For example, about 100 means between 90 and 110, preferably between 95 and 105.

“Consists of”, “consists essentially of” or “substantially comprises”: The description herein of any aspect of the description that uses terms such as reference to an element or elements is intended to provide support for a similar aspect of the description that “consists of,” “consists essentially of,” or “substantially comprises” that particular element or elements, unless otherwise stated or clearly contradicted by the context. For example, a peptide or protein described herein as comprising a particular sequence should be understood to also describe a peptide or protein consisting of that sequence, unless otherwise stated or clearly contradicted by the context. . By “consists essentially of” it is meant that the peptide or protein consists of that sequence, but may also include 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, additions or deletions. In particular, by "essentially consists of", it can be intended that the peptide may include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at the N or C terminus and 1, 2 or 3 substitutions, deletions or additions.

Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG or TTG and ends with a stop codon, such as TAA, TAG or TGA. The coding sequence may be genomic DNA, cDNA, synthetic DNA or a combination thereof.

Control Sequences: The term “control sequences” means nucleic acid sequences necessary for the expression of a polynucleotide encoding an epimerase of the present disclosure. Control sequences may be native (i.e., from the same gene) or heterologous (i.e., from a different gene and/or a different species) to the polynucleotide encoding the epimerase. Preferably, the control sequences are heterologous. Control sequences well known and currently used by those skilled in the art will be preferred. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, promoter, signal peptide sequence and transcription terminator. At a minimum, control sequences include a promoter and transcription and translation termination signals. The control sequences may be provided with linkers in order to introduce specific restriction sites that facilitate binding of the control sequences to the coding region of the polynucleotide encoding the epimerase. The functional combination of the control sequences and the coding sequences can be called an expression cassette.

Expression: The term “expression” includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.

Expression vector: The term “expression vector” means a linear or circular DNA molecule comprising a polynucleotide that encodes the epimerase of the description and is operatively linked to control sequences that provide for its expression. Therefore, the expression vector comprises an expression cassette suitable for expressing the epimerase of the description.

Isolated: The term “isolated” means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-natural substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, which is at least partially extracted of one or more or all of the natural constituents with which it is associated in nature; (3) any substance modified by man related to that substance found in nature; or (4) any modified substance by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a promoter stronger than the promoter naturally associated with the gene that codes for the substance).

Recombinant: Recombinant refers to a nucleic acid construct, a vector and a protein produced by genetic engineering.

Heterologous: In the context of a host cell, vector or nucleic acid construct, designates a coding sequence for the epimerase/peptide introduced into the host cell, vector or nucleic acid construct by genetic engineering. In the context of a host cell, it may mean that the coding sequence for the epimerase/peptide comes from a different source than the cell into which it is introduced. For example, an epimerase from Bacillus subtilis is expressed in E. coli. Alternatively, it may also mean that the coding sequence for the epimerase/peptide comes from the same species as the cell into which it is introduced, but is considered heterologous due to its unnatural environment, for example because it is under the control of a promoter that is not its natural promoter, or is introduced in a location that differs from its natural location.

Nucleic acid construct: The term “nucleic acid construct” means a nucleic acid molecule, whether single-stranded or double-stranded, that is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or that is synthetic, which comprises one or more control sequences.

Operationally linked: The term “operatively linked” means a configuration in which a control sequence is placed in an appropriate position with respect to a coding sequence, such that the control sequence directs the expression of the coding sequence.

Sequence identity: The sequence identity between two amino acid sequences is described by the parameter “sequence identity”. For the purposes of the present description, the "percent identity" between two amino acid sequences (A) and (B) is determined by comparing the two optimally aligned sequences, across a comparison window. Such sequence alignment can be carried out by well-known methods, for example, using the Needleman-Wunsch global alignment algorithm. The protein analysis software matches similar sequences using similarity measures mapped to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. Once the full alignment is obtained, the percent identity can be obtained by dividing the complete number of identical amino acid residues aligned by the total number of residues contained in the longest sequence between sequence (A) and (B).

Sequence identity is typically determined using sequence analysis software. To compare two amino acid sequences, one can use, for example, the “Emboss needle” tool for pairwise sequential alignment of proteins provided by EMBL-EBI and available at:

www.ebi.ac.uk/Tools/services/web/toolform.ebi?tool=emboss_needle&context=protein,

using default settings: (I) Array: BLOSUM62, (ii) Open Gap: 10, (iii) Gap Extent: 0.5, (iv) Output Format: Even, (v) Final Gap Penalty: False, (vi) final gap opening: 10, (vii) final gap extension: 0.5.

Alternatively, sequence identity can also typically be determined using Clustal Omega sequence analysis software using the HHalign algorithm and its default settings as the core alignment engine. The algorithm is described in Soding, J. (2005) 'Protein homology detection by HMM-HMM comparison'. Bioinformatics 21, 951-960, with default settings.

Amino Acids: The amino acid sequences defined herein are represented by a one-letter symbol as shown below: A, Ala, (alanine); R, Arg, (arginine); N, Asn, (asparagine); D, Asp, (aspartic acid); C, Cys, (cysteine); Q, Gln, (glutamine); E, Glu, (glutamic acid); G, Gly, (glycine); H, His, (histidine); I, Ile, (isoleucine); L, Leu, (leucine); K, Lys, (lysine); M, Met, (methionine); F, Phe, (phenylalanine); P, Pro, (proline); S, Ser, (serine); T, Thr, (threonine); W, Trp, (tryptophan); Y, Tyr, (tyrosine); and V, Val, (valine).

Conserved: Conserved amino acid means that a defined sequence aligns with the reference sequence and the remainder of the defined sequence corresponding to the position indicated in the reference sequence is identical to the remainder present in the reference sequence. The alignment can be carried out by any available method and, in particular, by the method disclosed for identity determination just above, more preferably by Clustal Omega. The position of the remainder is indicated in the reference sequence.

The term "antimicrobial" as used herein refers to antibacterial, antiviral, antifungal and/or antiparasitic activity. Said activity can be evaluated by measuring different parameters, such as IC ⁵⁰ or MIC.

“IC ⁵⁰ ” or “half-maximal inhibitory concentration” is the concentration of a substance necessary to reduce the in vitro growth of a population of microorganisms by half.

“MIC” or “minimum inhibitory concentration” is the lowest concentration of a substance that will completely inhibit microbial growth after 18 hours of incubation, generally at 37°C, in the presence of that substance.

The expression “lethal concentration, 50%” or “LC ^fifty ” as used herein refers to the concentration of substance required to kill half of a population. The CL ^fifty It is a quantitative indicator of the toxicity of a substance. In particular, the CL ^fifty is used herein to evaluate the cytolytic activity of AMP and, in this case, corresponds to the concentration of peptides that induces the lysis of half of the cell population.

Brief description of the figures

Figure 1. Purification, spectroscopic analysis, and activity of YydG. (Figure 1A) (Structure of the yydFGHIJ operon. yydF: putative peptide, yydG: radical SAM enzyme, yydH: protease, yydlJ ABC-type transporter. (Figure 1B) Gel electrophoresis analysis of purified YydG expressed in E. coli. (Figure 1B) Gel electrophoresis analysis of purified YydG expressed in E. coli . (Figure 1B) Figure 1C) UV-visible spectrum of anaerobically reconstituted YydG (Figure 1D) Multiple alignments of close YydF homologues found, in particular, in B. subtilis and Staphylococci species (Figure 1E) HPLC analysis of cleavage with SAM (257 nm ) and peptides (Figure 1F) produced in the reaction (280 nm) after 4 hours of incubation under anaerobic conditions in the presence or absence of sodium dithionite (2 mM). (Figure 1G) Time course for the production of 5' -dA (■, and modified peptides (♦) quantified by reverse-phase HPLC and monitored by UV-visible detection (280 nm). YydG (100 μM) was incubated under anaerobic conditions with sodium dithionite (2 mM) in the presence of 1 mM YydF substrate i8-49 .(Figure 1H and I) HPLC analysis of YydG incubated with (Figure 1H) YydF or (Figure 1I) a truncated version of YydF18-49. YydG, after anaerobic reconstitution, was incubated under anaerobic conditions in the presence of DTT (6 mM) and SAM (1 mM) with or without sodium dithionite (2 mM).

Figure 2 - YydG catalyzes the transfer of H atom to the peptide backbone. Tryptic peptide mapping and LC-MS analysis of (Figure 2A) YydF 18-49 or (Figure 2B) YydF 18-49 following incubation with YydG. The numbers indicate the m/z value for each peptide. The sequence in bold indicates the relevant peptide identified by LC-MS (i.e., Peptide 1: NH ² -GLLDESQK, [M+H] + = 931.48; Peptide 2: VNDLWYF [M+2H] 2+ =592 .31; Peptide 3: W IlGs GH-Ac, [M+H] + = 768.41). (Figure 2C) LC-MS analysis of YydF 18-49 peptide after incubation with YydG in deuterated buffer. (Figure 2D) Tryptic peptide mapping and LC-MS analysis of YydF 18-49 after incubation with YydG in deuterated buffer.

Figure 3 - YydG catalyzes amino acid epimerization. LC-MS/MS analysis of a (Figure 3A) L-Ile/D-allo-Ile and (Figure 3B) L-/D-Val (upper traces) in comparison with the amino acids obtained after incubation of YydF 18- 49 with the rSAM enzyme YydG in deuterated buffer (lower traces). Amino acids were derived by Wa-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA) and detected by LC-MS as FDVA derivatives.

Figure 4 - Activity of YydG mutants. (Figure 4A) Sequence of YydG with the cysteine residues highlighted. (Figure 4B) Gel electrophoresis analysis of the purified mutant enzymes. (Figure 4C) UV-visible spectra of A3 (i.e. AxxxAxxA) (blue trace), C ₂ 2A (green trace), C ₂ 22A (red trace) and C ₂ 23A (purple trace) mutants after anaerobic reconstitution . (Figure 4D) HPLC analysis of the reaction after incubation of YydF18-49 and the four mutants in the presence of SAM (1 mM) and sodium dithionite. (Figure 4E) LC-MS analysis of the peptides produced by the C ₂ 23A mutant and their corresponding masses and sequences.

Figure 5 - YydF18-49 activity of YydG. (Figure 5A) Growth inhibition assay on B. subtilis plates in the presence of YydFis-49 or the epimerized product YydFb. (Figure 5B) Growth of Bacillus subtilis in LB medium in the absence of peptide (□) or in the presence of the YydFis-49 peptide with a free C terminus: Ac-GLLDESQKLAKVNDLWYFVKSKENRWILGSGH (SEQ ID NO:20) containing no modification (■), a D-allo-Ile (♦), one D-Val (O) or two epimerized residues: Ac-GLLDESQKLAKVNDLWYF{d-V}KSKENRW{d-l}LGSGH (SEQ ID NO: 20) (A). OD values are the means of 3 independent cultures.

Figure 6- Multiple alignments of close homologs of YydG found, in particular, in B. subtilis and staphylococcus species.

Figure 7 - Mass spectrometry analysis of the YydF peptide isolated from B. subtilis and called YydF 33-49 . LC-MS/MS analysis of (a) the peptide secreted by B. subtilis, (b) synthetic YydF33-49DD peptide containing two D-amino acid residues, and (c) synthetic YydF33-49 peptide. Relevant ions are indicated. Tables 1 for the full assignment. In the peptide sequence, amino acids with a D configuration are indicated in gray.

Figure 8 -(A) Growth of B. subtilis in liquid LB medium in the presence of YydF ^{33^9} or YydF33^9DD. B. subtilis was cultured in LB medium alone (♦), in the presence of YydF33-49(ü) or YydF33-49DD (■). Each measurement is the mean of three growth experiments with the indicated SD. Epimerized remains are in bold. (b) Growth rate of B. subtilis in the presence of YydF18-49 (100 μM) or YydF18-49DD (100, 10, or 1 μM). The ratios were determined by comparison with growth in the absence of peptide. (c) Growth ratio of B. subtilis in the presence of YydF33-49 or YydF33-49DD (100, 10, 1.0.1 or 0.01 μM). The ratios were determined by comparison with bacterial growth in the absence of peptide.

Figure 9 - Growth of B. subtilis in liquid LB medium in the presence of YydFis-49 (□) or after the addition (Arrow) of YydFi8-49DD (■) after 3 hours of growth. Each measurement is the mean of three growth experiments with the indicated SD.

Figure 10 - Growth of B. subtilis in liquid LB medium in the presence of YydF33-49 (a), YydF33_49AA (b), YydF33_49w (c), YydF33_49II (d), Peptide-SA1 (e), Peptide-SA2 (f) , Peptide-SE (g) or Peptide-SP (h) at 100 μM. In bold, epimerized remains.

Figure 11 - Growth of Enterococcus faecalis in liquid BHI medium in the presence of YydF33-49 (a), YydF33_49AA (b), YydF33_49w (c), YydF33_49ii (d), Peptide-SA1 (e), Peptide-SA2 (f), Peptide-SE (g) or Peptide-SP (h) at 100 μM. In bold, epimerized remains.

Figure 12 - Growth of Streptococcus agalactiae in liquid BHI medium in the presence of YydF33-49 (a), YydF33_49AA (b), YydF33_49w (c), YydF33_49ii (d), Peptide-SA1 (e), Peptide-SA2 (f), Peptide-SE (g) or Peptide-SP (h) at 100 μM. In bold, epimerized remains.

EXAMPLES

Example 1

The inventors herein showed that the common laboratory strain Bacillus subtilis is capable of producing a new type of bioactive peptides containing D-amino acids despite being of ribosomal origin. This peptide is post-translationally modified by a new enzyme that belongs to the radical SAM superfamily of enzymes. They showed that this new enzyme uses an unprecedented radical-based mechanism to convert L-isoleucine and L-valine residues to D-allo-isoleucine and D-valine. They established that this enzyme generates a 5'-deoxyadenosyl radical to catalyze the abstraction of the H atom from Ca leading to the formation of a carbon-centered radical. Mutagenesis experiments support that this enzyme has two essential centers [4Fe-4S] and allow the identification of a critical H atom donor, necessary for the completion of the catalytic cycle. Finally, in a unique way, they discovered that the presence of D-amino acids is necessary for the activity of this bioactive peptide that likely induces LiaRS, a major component of bacterial wall integrity.

The Lia system from Bacillus subtilis is a cell envelope stress module composed of a two-component system (LiaRS) and an inhibitory protein (LiaF). This genetic system is highly conserved among Firmicutes and part of the complex regulatory network that orchestrates the cell wall stress response. Although its regulation has been described in great detail, its precise physiological role in B. subtilis is not fully understood. LiaRS is specifically and strongly induced by cell wall-targeting antibiotics such as nisin, vancomycin or bacitracin and has therefore been developed as a high-throughput biosensor and screen for cell wall antibiotics. Upon detection with antibiotics, LiaRS transduces cell envelope stress signals that activate gene expression, presumably to maintain cell wall integrity even though it does not confer antibiotic resistance.

In an attempt to identify the genes involved in the regulation of LiaRS, a mutagenesis study was performed in B. subtilis and led to the discovery of the yydFGHIJ operon (Butcher et al, 2007, J Bacteriol, 189 , 8616). This operon shows positive regulation in LiaRS and possesses all the characteristic features of a genetic system that encodes a putative peptide (YydF) modified by a radical SAM enzyme and a protease (YydG and YydH respectively), then finally exported into the extracellular medium. by an ABC-type transporter (YydlJ) despite the fact that none of these components were isolated or investigated (Figure 1A) .

Radical SAM enzymes are an emerging family of enzymes that catalyze a wide diversity of protein and peptide modifications, such as oxidation, unusual methyl transfer reaction, or thioether bond formation. They have emerged as major players in the biosynthesis of so-called RiPPs (ribosome-synthesized and post-translationally modified peptides) that are involved in chemically challenging reactions that no other enzyme can perform. To investigate the biological role and catalytic function of the putative radical SAM enzyme YydG, the inventors overexpressed the protein in E. coli and tested its activity against the YydF peptide. The purified protein (Figure 1B) had the distinctive spectroscopic properties of radical SAM enzyme with charge transfer absorption band at 320 and 420 nm (Figure 1C). Based on a molar extinction coefficient of the [4Fe-4S]2+ cluster of £ ⁴¹⁰ ~15,000 M'1 cm-1, the enzyme appears to possess one to two [4Fe-4S] centers after anaerobic reconstitution (Figure 1C ).

Genome mining revealed that yydF and yydG are also present in several Gram-positive pathogens, such as Enterococcus faecalis and various Streptococci and Staphylococci, including S. agalactiae and S. epidermidis. Sequence alignment of YydF homologs indicated a putative leader sequence located in the N-terminal part and a highly conserved motif from residue 17 to the end of the peptide (Figure 1D). We tested the reconstituted enzyme with either the full-length YydF peptide or a truncated form, spanning the conserved amino acid residues from position 18 to 49, which we termed YydF18-49. As shown (Figure 1E), under anaerobic conditions and reducing conditions, YydG produced the predicted 5'-deoxyadenosine (5'-dA; eluting at 12.3 min) resulting from the homolytic cleavage of S-adenosyl-L-methionine ( SAM) and also three peptide derivatives: YydFa, YydFb and YydFc eluting at 46, 47.6 and 48 min (Figure 1E, F, G and H). The formation of the three peptides was strictly dependent on the presence of sodium dithionite as an electron donor and similar products were obtained using YydF or YydF18-49 (Figure 1F, G and H). Kinetic analysis of the reaction showed that YydG produced one mole of 5'-dA per mole of modified product and catalyzed several returns under in vitro conditions, although uncoupled SAM cleavage occurred as the reaction proceeded (Figure 1G). These results demonstrated that, in vitro, YydG utilized SAM to modify YydF through a radical-based mechanism. Since YydF18-49 was shown to be a better substrate and easier to characterize, the inventors decided to identify the modification catalyzed by YydG.

Mass spectrometry inspection of the three peptides formed did not reveal any mass differences compared to the substrate YydF18-49 [M+3H]3+= 1258.92). This was in contrast to all known rSAM enzymes that catalyze posttranslational modifications of peptides or proteins, such as cross-linking, oxidation, or methylation (7, 9, 14-16). Tryptic peptide mapping of the substrate gave three peptides ( Peptide 1 [M+H]+ =931.48, Peptide 2 [M+H]2+ =592.31 and Peptide 3 : [M+H]+= 768, 41) eluting at 22, 27 and 19.2 min) as shown in Figure 2A . Comparison with the enzymatically modified peptide showed the appearance of two new peptides (i.e., Peptide 2* and Peptide 3* ) that have the same molecular weight as Peptides 2 and 3 but elute at 26.5 and 23, 5 min, respectively (Figure 2B). This result supported that YydG had introduced two modifications, one located internally (in Peptide 2 ) and one at the C-terminal end of the peptide (in Peptide 3 ). In all experiments carried out, YydFc was the main product. Tryptic peptide mapping revealed that essentially the C-terminus of the peptide had been modified, as the Peptide 3*/Peptide 3 ratio was 5-fold greater than the Peptide 2*/Peptide 2 ratio (Figure 2B) , indicating that YydG had some preferred sites.

To identify the nature and location of the modifications catalyzed by YydG, the inventors repeated the reaction in >90% deuterated buffer, since rSAM enzymes are known to subtract, and sometimes exchange, H atoms during catalysis. In deuterated buffer, YydG produced a similar product pattern with YydFc always being the most abundant product (Figure 2C). Interestingly, LC-MS analysis of the reaction showed that, under these conditions, YydFa and YydFc had a molecular weight of [M+3H]3+= 1259.24 and YydFb a molecular weight of [M+3H]3+= 1259.6. This corresponded to one and two Dalton units more than the substrate, YydF18-49 ([M+3H]3+= 1258.92), unequivocally, demonstrating that one deuterium atom was incorporated into YydFa, YydFc and two deuterium atoms in YydFb, respectively. Tryptic peptide mapping of the reaction allowed deuterium incorporation to be located exclusively in Peptide 2* and Peptide 3* whose molecular masses varied from one Dalton unit (i.e., [M+2H]2+ = 593.04 and [M+2H]+= 769.52) (Figure 2D). Fragmentation of these two peptides demonstrated deuterium incorporation at Val 36 in Peptide 2* (as shown by the characteristic ions y ¹ , y2+1 and b7, b8+1) and at Ile 44 in Peptide 3* (as shown by identifying the ions ys and y6+1). Altogether, these results demonstrated that YydG catalyzes the substitution of two H atoms of the peptides by two exchangeable H atoms of the solvent.

To determine the nature of the modification, the inventors performed acid hydrolysis of the peptide and analyzed its amino acid content, after derivatization with Wa-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA). , by LC-MS. The YydF18-49 peptide contains two Val residues and one Ile residue, but five Leu residues that not only have the same molecular weight as Ile but also elute at similar retention times. The optimized LC-MS/MS conditions allowed, as shown in Figure 3A, the separation and characterization of L-lle and L-Leu, but also their counterparts with D configuration (D-allo-lle and D-Leu ). Analysis of the enzymatically modified peptides clearly showed that, in addition to the identification of L-Ile and L-Leu, another product was formed that eluted at 27.7 minutes, corresponding to D-allo-Ile. Likewise, the analysis of the Val residues (Figure 3B) showed the presence of not only L-Val but also D-Val, eluting at 26 min.

Therefore, the inventors established that YydG is a radical SAM epimerase, the first to be shown to be active in vitro on a peptide structure. The enzyme catalyzed the epimerization of up to ~35% Ile and ~10% Val residues. Consistent with this conclusion, when epimerized amino acids were derivatized by incubating YydG in deuterated buffer, their mass analyzes revealed an increase of 1 Da (Figure 3C and D) , consistent with the analyzes performed on the intact peptide and its tryptic derivatives (Figure 2C and D).

To definitively affirm their identity as D-configuration amino acids, the inventors synthesized a variant peptide of YydF18-49 containing a D-Val and a D-allo-lle at positions 36 and 44, respectively. The tryptic peptide mapping and amino acid profile of this synthetic peptide perfectly reproduced those of the enzymatically modified peptide (data not shown).

Based on these analyses, the inventors were able to assign YydFa as a peptide containing a D-Val at position 36, YydFc as a peptide containing a D-allo-lle at position 44, and YydFb as a peptide containing a D-Val. Val and a D-allo-lle in positions at positions 36 and 44, respectively. YydG, therefore, produced a mixture of peptides containing single or double modified amino acids, with Ile44 being the preferred substrate (Figure 2C).

During the epimerization reaction performed in deuterated buffer, if the inventors have established that a solvent-exchangeable H atom is incorporated into the peptide backbone, the 5'-dA produced did not contain significant labeling as shown by LC-analysis. M. These results and the kinetic analysis in Figure 1G are consistent with YydG producing a 5'-dA radical (5'-dA^) to abstract the Ca H atom from Val36 or lle44 with concomitant formation of one mole of 5 '-gives.

The last questions that remained to be resolved were the origin of the exchangeable H atom introduced during catalysis. In fact, the carbon-centered radical was unlikely to interact with a buffer component, since such highly reactive species must remain sealed in the active site of the enzyme. The inventors favored a protein amino acid residue as the donor of H atoms and a radical quencher was required to terminate the reaction. Close inspection of the YydG sequence noted that, in addition to the three cysteine residues of the radical SAM motif, only six cysteines were present in the sequence (Figure 4A).

Interestingly, two cysteine residues (i.e., Cys22 and Cys223 ) were adjacent to another cysteine residue, one of which was within the predicted loop containing the rSAM [4Fe-4S] center. The organization of the other five cysteine residues at the C-terminus of the protein was reminiscent of the motifs involved in the coordination of the additional [4Fe-4S] centers in rSAM enzymes. To investigate its function, the inventors replaced the cyanine residues of the radical SAM motif CxxxCxxC, Cys22, Cys222 or Cys223 with alanine residues. The four designed mutants (i.e., A3, C ₂ 2A, C ₂ 22A, and C ₂ 23A ) were successfully purified. although the C ₂ 22A mutant proved to be recalcitrant to purification and was partially produced as a truncated form (Figure 4B) . Spectroscopic analysis showed that, based on its UV-visible spectrum, the AxxxAxxA mutant contained ~1 [4Fe-4S] center, while the amount of [4Fe-4S] center was twofold higher in the C ₂ 2A mutants. and C ₂ 23A (Figure 4C) . Importantly, the aerobically purified AxxxAxxA mutant already contained high amounts of iron-sulfur center, demonstrating that the presence of the [4Fe-4S] center in this mutant was independent of anaerobic reconstitution. The UV-visible spectra of the C ₂ 2A and C ₂ 23A mutants overlap perfectly with the wild-type enzyme ( Figure 1B) , confirming the fact that YydG likely contains two [4Fe-4S] centers. The C ₂ 22A mutant appeared to contain no iron-sulfur center, even after anaerobic reconstitution (Figure 4C ). The absorption maximum of C ₂ 22A shifted towards 250 nm, indicating that the protein was probably misfolded, as it has been repeatedly reported when cysteine residues involved in [4Fe-4S] coordination are mutated in enzymes. with iron-sulfur, including rSAM enzymes. Therefore, Cys222 is likely involved in the coordination of the second [4Fe-4S] center present in YydG.

The activity of all mutants was tested with the substrate YydF18-49 (Figure 4D). As expected, the A3 mutant was unable to convert the peptide substrate and cleave SAM. The C ₂ 22A mutant was also completely impaired in enzymatic activity. In contrast, the C ₂ 2A mutation did not affect the activity of the enzyme and all three epimerized peptides (i.e., YydFa, YydFb, and YydFc) were produced. The C ₂ 23A mutation also did not prevent epimerization activity. However, this enzyme variant produced other peptide derivatives that eluted at 10 min, 19.8 min, and 26 min (Figure 4E). High-resolution mass spectrometry showed that these peptides have a mass shift of -30.005 Da or -1.032 Da, compared to the predicted hydrolytic products. All of them contained at their C or N terminus a truncated Val36 or Ile44, the targets of the YydG enzyme (Figure 4 ^e ). Its structure was determined as: Ac-GLLDESQKLAKVNDLWYFVKSKENRWI* (SEQ ID NO: 53) (peptide G ¹⁸ -I ⁴⁴ *, [M+2H]2+= 1646.3834); and l *LGSGH-NH ² (SEQ ID NO: 55) (peptide G ¹⁸ -I ⁴⁴ *, [M+Na]+= 603.2851). Truncation was identified as the loss of the carboxylic amino acid or amino group, as a result of the cleavage of the C aN or Ca-CO bonds, and the addition of an oxygen atom to the Ca atom of the amino acid. These results are reminiscent of the substrate cleavage obtained with another member of the rSAM enzyme family, pyruvate formate lyase activase, when the reaction was exposed to molecular oxygen. They also definitively established that YydG generates a carbon-centered radical at the Ca atom of Val36 and Ile44 and that Cys223 plays a critical role for the termination of the reaction.

In light of the inventors' previous work on another rSAM enzyme, spore photoproduct lyase (SP lyase), the inventors interpreted the role of Cys223 as a critical H atom donor. In fact, while investigating a mutant of the S ^p lyase, have shown that, in the absence of a suitable protein H atom donor, the radical intermediate substrate can react with adventitious radical scavengers leading to the formation of various adducts. In the present case, the stabilized Ca radical, in the absence of the thiol group of Cys223, is free to react with molecular oxygen, leading to these unique breaks of the peptidyl main chain.

Finally, since the YydFGHIJ operon (Figure 1A) was shown to activate the Lia system in B. subtilis, the inventors tested the activity of the YydF18-49 peptide before and after the enzymatic reaction in various bacterial strains. They revealed strong growth inhibition of B. subtilis with the enzymatically epimerized peptide or the synthetic peptide containing D-Val36 and D-allo-Ile44. In contrast, the unmodified YydF18-49 peptide lacked activity (Figure 5A) . Likewise, in liquid medium, only the epimerized peptide proved to be active, since a strong and persistent inhibition could be measured (Figure 5B). More studies will be needed to decipher the molecular basis of this inhibition, but to the inventors' knowledge, this is the first time that a naturally epimerized peptide was shown to be the active form of a regulatory or antimicrobial bacterial peptide.

The present study demonstrates that peptides containing D-amino acids, called Epipeptides, are much more common than expected in living organisms, including the common laboratory bacterium Bacillus subtilis, but also many pathogenic species such as Streptococcus agalactiae, Enterococcus faecails or Staphylococcus epidermidis. Unexpectedly, the inventors demonstrated in this case that D-amino acids appear not only to provide resistance to proteases but are directly involved in the bacterial response.

Example 2

It has been proposed in the literature that a gene, yydF encodes a peptide produced by Bacillus subtilis (SEQ ID NO: 1). However, no evidence of its actual synthesis or any post-translational modification has been reported.

After growth of B. subtilis on synthetic medium (5X buffer solution (Na ₂ HP04: 17 g; KH ₂ P04: 7.5 g, NaCl: 1.25 g; NH4CI: 2.5 g in 500 ml); Trace element solution: MnCh: 20 mg; ZnCh: 34 mg; CuCh: 8.6 mg; CoCh: 12 mg; Na2MoO ₄ : 12 mg; in 200 ml), the inventors successfully purified a peptide, which originates from of YydF and spanning residues 33 to 49 (Figure 7), established by mass spectrometry analysis (Table 1). The sequence of the isolated peptide was determined to be (SEQ ID NO:61):

Trp Tyr Phe Val Lys Ser Lys Glu Asn Arg Trp lie Leu Gly Ser Gly His

SEQ ID NO: 61: YydF 33_49 peptide sequence

Furthermore, the inventors determined that the YydF33-49 peptide, produced by B. subtilis, contained 2 epimerized residues (i.e., D-amino acids) located at position 36 (Val) and 44 (Ile). Therefore, the peptide was named YydF_ 33-49DD. The inventors' previous work (Example 1) established that the epimerized residues are the result of the conversion of L-amino acid residues by a single radical SAM enzyme, YydG, which targets the Ca atom of the amino acids. At present, it is not known whether such short peptides, containing discrete epimerization, are produced by bacteria.

Because the YydFGHIJ operon was shown to induce the two-component system LiaRS, which, among other stimuli, sensed the integrity of the bacterial cell wall, the inventors searched for a putative inhibition of bacterial growth triggered by various derivatives of the YydF peptide. Initial tests were performed with a peptide spanning residues 18-49 (YydF18-49, SEQ ID NO:20 ).

As shown (Figure 5B), only in the presence of the YydF18-49DD peptide containing two epimerized residues: Val36 and Ile44 (numbering according to SEQ ID NO: 61), we monitored the inhibition of bacterial growth. The presence of an epimerized residue or the absence of epimerized residues did not significantly affect bacterial growth. This demonstrated, for the first time, that a short peptide with epimerized residues can inhibit bacterial growth.

Having established that the presence of two key epimerized residues is crucial for the inhibitory properties, the inventors synthesized two peptides corresponding to the sequence of the peptide produced by B. subtilis (SEQ ID NO: 61). These two peptides contained either only L-amino acid residues (YydF33-49) or the two crucial epimerized residues: D-Val36 and D-Ile44 (YydF33-49DD). Only the YydF33-49DD peptide was shown to inhibit bacterial growth (Figure 8a) . This new peptide was shown to be 100 times more potent than the previously tested YydF18-49DD peptide (Figure 8b and c) with an MIC ≤1 μM. Furthermore, the inventors demonstrated that these peptides not only inhibited bacterial growth but also induced bacterial cell death. In fact, if after an initial growth of 3 hours, the YydF18-49 peptide is added in the mid-exponential phase (Figure 9) , a clear slowdown was measured, followed by a decrease in cell density.

Interestingly, homologs of YydF peptides are predicted in the genome of several Gram-positive bacteria, such as: Salinibacillus aidingensis, Bacillus coagulans, Paenibacillus sp and several pathogenic species, such as: Enterococcus faecalis, Enterococcus caccae, Streptococcus agalactiae, Staphylococcus pseudintermedius, Staphylococcus equorum, Staphylococcus condimenti and Staphylococcus epidermidis (Figure 1D, Figure 6) .

In order to determine whether these peptides are bioactive, the inventors synthesized a library of peptides based on sequences identified in the genomes of Streptococcus and Staphylococcus species. They hypothesized that these peptides should contain the same post-translational modifications as those identified in B. subtilis, meaning a processed peptide of 17 amino acid residues with two D-amino acids at positions 4 and 12 (SEQ ID NO: 62) . -65) . Epimerized remains are in bold.

Streptococcus agalactiae Peptide-SA1 WYFVRSSKNRWVAGSAH (SEQ ID NO: 62)

Streptococcus agalactiae Peptide-SA2 WYFVRNSKNRWVAGSAH (SEQ ID NO: 63)

Staphylococcus equorum Peptide-SE WYFVKSKQNRWWGSGH (SEQ ID NO: 64)

Staphylococcus pseudintermedius Peptide-SP WYFVKSQSNRWIVGSGH (SEQ ID NO: 65)

In addition, the inventors also synthesized three unnatural peptides derived from the YydF33_49 sequence of B. subtilis (SEQ ID NO: 61), but for which the two epimerized residues (i.e., Val36 and Ile44) were both replaced by residues of Val, Ile or Ala, YydF33_49VV, YydF33_49II and YydF33_49AA, respectively (SEQ ID NOS 66-68). Epimerized remains are in bold.

^YydF33_49AA WYFAKSKENRWALGSGH ^{(SECIDn.°66)}

YydF33_49VV WYFVKSKENRWVLGSGH (SEQIDn°67)

YydF33_49ii WYFIKSKENRWILGSGH (SEQ ID No. 68)

These 7 peptides (SEQ ID NOs 62-68) were tested against B. subtilis and the two representative Gram-positive pathogens: S. agalactiae and E. faecalis. As shown, all peptides were effective against B. subtilis, including peptides with unnatural sequences (Figure 10). The growth of E. faecalis was significantly delayed by Peptide-SA1 and Peptide-SA2, and growth was totally inhibited by Peptide-SE and the unnatural peptide YydF33_49AA (Figure 11). S. agalactiae was inhibited by YydF33-49DD and the derivatives YydF33-49AA and YydF33-49w but not by YydF33-49II (Figure 12). The peptides: Peptide-SA1, Peptide-SA2, Peptide-SE and Peptide-SP were all inhibitors.

The inventors thus demonstrated that short peptides containing two D-amino acid residues are a new class of inhibitory peptides capable of inhibiting the growth of various Gram-positive bacteria, including relevant pathogens. They are effective if added at the beginning or after bacterial growth in the middle of the exponential phase. Finally, some small modifications in the sequence are capable of adjusting the inhibition properties and specific characteristics at the genus and species level of these peptides, thus allowing the development of specific antibiotics. Furthermore, based on the 17 amino acid framework and the conserved location of two D-amino acids (at position 4 and 12) downstream of the aromatic residues (W or Y), the inventors also demonstrated that it is possible to design peptides with sequences not natural that proved to be effective against all gram-positive bacteria analyzed.

The bioactive peptides were shown to have varying sequence identity between 100 and 58.8% relative to the original YydF33-49 sequence meaning that at least 7 amino acid residues could be changed without altering their overall inhibition properties. It is therefore possible to design these peptides in an unprecedented way to target specific bacterial genera and tune their biological properties.

No. Percentage Identity Matrix No.

1 Peptide-SA1 100.00 94.12 64.71 70.59 58.82 58.82 64.71 70.59 2 Peptide-SA2 94.12 100.00 58.82 64.71 52.94 52.94 58.82 64.71 3 Peptide-SP 64.71 58.82 100.00 82.35 70.59 76.47 82.35 76.47 4 Peptide-SE 70.59 64.71 82.35 100.00 76.47 76.47 82.35 88.24 5 YydF33 49AA 58.82 52.94 70.59 76.47 100.00 88.24 88.24 88.24 6 YydF33 49II 58.82 52.94 76. 47 76.47 88.24 100.00 94.12 88.24 7: YydF33_49DD 64.71 58.82 82.35 82.35 88.24 94.12 100.00 94.12 8: YydF33_49VV 70.59 64 .71 76.47 88.24 88.24 88.24 94.12 100.00 Materials and procedures

YydG expression

The yydG genes were synthesized (Life Technologies) and cloned into a pASK plasmid. The plasmid was expressed in E. coli BL21 (DE3) star (Life Technologies) and protein expression was performed in LB medium with ampicillin (100 μg.ml-1). After growing overnight at 21°C, cells were harvested and disrupted by sonication in buffer A (50 mM Tris, 300 mM KCl, 10% glycerol pH 7.5). The bacterial suspension was centrifuged at 45,000 x g for 1.5 hours and the protein supernatant was loaded onto a Streptactin high capacity column (IBA GmbH) previously equilibrated with buffer A. The YydG protein was eluted with 6 ml of buffer A containing Desthiobiotin (0.6 mg/ml) further concentrated with the Amicon concentrator (Millipore) with a molecular cutoff value of 10 kDa.

Enzyme reconstitution

YydG was reconstituted under anaerobic conditions in a Bactron IV anaerobic chamber. The protein was mixed with 3 mM DTT at 12 °C for 15 minutes, then Na ² S and (NH4) ₂ Fe(SÜ4) ₂ were added and the solution was incubated at 12 °C for 4 hours.

Enzymatic assays

YydG was incubated with 3 mM DTT, 1 mM SAM, and 1 mM peptide substrate unless otherwise indicated. Incubations were performed at 25°C under strict anaerobic conditions and 10 μl aliquots were sampled over time.

HPLC analysis

HPLC analysis was performed on Agilent 1200 infinity series equipped with a reversed-phase column (LiChroSphere 100 RP-18e 5 μm) (Merck Millipore). A gradient from solvent A (H ² O, 0.1% TFA) to B (80% CH ³ CN, 19.9% H ² O, 0.1% TFA) was applied as follows: 0-1 min: 100% N 0% B; 1-45 min: a linear gradient with 1% solvent B per minute at a flow rate of 1 ml.min-1. Detection was performed at 257 and 278 nm with a diode array detector and by fluorescence (excitation at 278 nm and emission at 350 nm).

Analysis by liquid chromatography-mass spectrometry/mass spectrometry

High-performance liquid chromatography-mass spectrometry/mass spectrometry analyzes were performed using an LTQ-Orbitrap Discovery mass spectrometer (ThermoFisher) with a nanoelectrospray ion source and an Ultimate 3000 LC system (Dionex). An LTQ mass spectrometer (ThermoFisher) with a nanoelectrospray ion source was used for routine analysis. Peptide analysis was performed on a Pepmap 100 C18 nanocolumn (0.075 by 15 cm, 100 A, 3 μm).

Inhibition assay in solid medium

An overnight culture of the bacterial strain for testing was inoculated freshly into sterile BHI liquid medium. After 4 h of bacterial growth at 37°C, the medium was diluted 1/1000 and inoculated into soft agarose medium prewarmed to 42°C. The agarose medium containing the bacteria was overlaid on a layer of previously gelled sterile BHI agarose. 200 μg of peptide was placed on the plate and bacterial growth proceeded at 37 °C.

Inhibition assay in liquid medium

An overnight culture of the bacterial strain for testing was inoculated freshly into sterile LB liquid medium. After 4 hours of bacterial growth at 37°C, the medium was diluted to 1/10,000 and inoculated into sterile liquid LB or BHI medium. Peptide solution (1/100) was added to a final concentration ranging from 0.01 to 100 μM and an OD at 600 nm was recorded continuously using a Tecan microplate reader (Infinite® 200 PRO series). Table 1

Mass fragments for the YydF33-49 peptide isolated from B. subtilis

Claims (18)

1. A peptide having antibacterial activity having at least two amino acids in D configuration, comprising a sequence of 17 residues having at least 40% identity with the sequence at positions 33 to 49 of SEQ ID NO: 1 and said sequence comprises the sequences W-Y-F-[V/I/A] (SEQ ID NO: 71) and W-[lN/A]-X4-G-S (SEQ ID NO: 69), wherein X4 is any amino acid, and a junction sequence between SEQ ID NO:69 and SEQ ID NO:71 of 4-8 amino acids, in which residues [V/I/A] of SEQ ID NO:69 and 71 have a D configuration and where the peptide has a length of about 17 to about 35 amino acids. 2. The peptide according to claim 1, wherein the peptide comprises a sequence of W-Y-F-[V/I/A]-[K/R]-Xa-Xb-Xc-N-R-W-[V/I/A]- Xd-G-S-Xe-H (SEQ ID NO: 72), wherein Xa, Xb and Xc are a polar amino acid, Xd is an aliphatic amino acid and 3. The peptide according to claim 2, wherein the peptide comprises a sequence of W-Y-F-[V/I/A]-[K/R]-[S/N]-[S/Q/K]-[E /K/S/Q]-N-R-W-[V/I/A]-[LN/A]-G-S-[NG]-H (SEQ ID NO: 73). 4. The peptide according to any one of claims 1-3, wherein the peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 61-68. 5. The peptide according to claim 1, wherein the peptide comprises a sequence of 24 residues that has at least 40% identity with the sequence at positions 26 to 49 of SEQ ID NO: 1 and said sequence comprising the sequences N/D-D-L-W-Y-F-[V/I/A] (SEQ ID NO: 21) and W-[lN/A]-X4-G-S (SEQ ID NO: 69), wherein X4 is any amino acid and a junction sequence between SEQ ID NO: 21 and SEQ ID NO: 69 of 4-7 amino acids and wherein the peptide has a length of about 25 to about 35 amino acids. 6. The peptide according to claim 5, wherein the peptide comprises the sequences of L-X1-X2-X3-N-D-L-W-Y-F-V/I (SEQ ID NO: 23) and W-IN-X4-G-S (SEQ ID NO: .° 22), in which X1, X2, X3 and X4 are any amino acid. 7. The peptide according to claim 6, wherein X1 is alanine, aspartic acid or glutamic acid, X2 is lysine, asparagine or aspartic acid and X3 is valine, isoleucine or glutamine. 8. The peptide according to claim 5, wherein the peptide comprises the sequences L-A-K-V-N-D-L-W-Y-F-V (SEQ ID NO: 24), and W-IN-X4-G-S (SEQ ID NO: 22), wherein X4 is a hydrophobic amino acid, preferably leucine, valine, alanine or methionine. 9. The peptide according to any one of claims 5-8, wherein the peptide comprises, consists essentially of or consists of the sequence of SEQ ID NO: 20 and the valine at position 19 is in D configuration and the isoleucine in position 27 it is in configuration D. 10. A pharmaceutical composition comprising a peptide according to any one of claims 1-9. 11. The peptide according to any one of claims 1-9 for use as a drug, preferably as an antimicrobial, more preferably as an antibacterial. 12. An in vitro method for preparing a peptide according to any one of claims 1-9, wherein the method comprises a step of contacting a peptide comprising a sequence of 17 residues having at least 40% identity. with the sequence at positions 33 to 49 of SEQ ID NO: 1 and said sequence comprises the sequences WYF-[V/I/A] (SEQ ID NO: 71) and W-[l N /A]- X4-GS (SEQ ID NO: 69), wherein has a length of about 17 to about 35 amino acids, with an epimerase having an amino acid sequence that has at least 30% identity with SEQ ID NO: 25 and in which 70% of positions 14-15 , 18, 20-24, 27, 58-60, 63, 83, 84, 87-90, 112, 115, 117, 120, 150, 152, 169, 176, 180, 181, 183, 204, 206-208 , 214, 217, 220-225, 228, 230, 252, 254, 262, 289, 292, 296 and 309 of SEQ ID NO: 25 are preserved and all positions 14-15, 18, 20-24, 27, 58-60, 63, 83, 84, 87-90, 112, 115, 117, 120, 204, 206-208, 214, 217, 220 225, 228 and 230 of SEQ ID NO: 25 are retained . 13. The in vitro method of claim 12, wherein the peptide comprises a 24-residue sequence having at least 40% identity with the sequence at positions 26 to 49 of SEQ ID NO: 1 and said sequence comprises the sequences N/DDLWYFV/I (SEQ ID NO: 21) and W-lN-X4-GS (SEQ ID NO: 22), wherein X4 is any amino acid and a junction sequence between SEQ ID NO: 21 and SEQ ID NO: 22 of 4-7 amino acids, and wherein the peptide has a length of about 25 to about 35 amino acids. 14. Use of a radical SAM peptide epimerase having an amino acid sequence that has at least 30% identity with SEQ ID NO: 25 and in which 70% of positions 14-15, 18, 20 -24, 27, 58-60, 63, 83, 84, 87-90, 112, 115, 117, 120, 150, 152, 169, 176, 180, 181, 183, 204, 206-208, 214, 217 , 220-225, 228, 230, 252, 254, 262, 289, 292, 296, and 309 of SEQ ID NO: 25 are preserved and all positions 14-15, 18, 20-24, 27, 58 -60, 63, 83, 84, 87-90, 112, 115, 117, 120, 204, 206-208, 214, 217, 220-225, 228 and 230 of SEQ ID NO: 25 are conserved to prepare a peptide according to any one of claims 1-9. 15. A recombinant host cell comprising a heterologous nucleic acid encoding a peptide comprising a 17-residue sequence having at least 40% identity with the sequence at positions 33 to 49 of SEQ ID NO: 1 and comprising the sequences W-Y-F-[V/I/A] (SEQ ID NO: 71) and W-[lN/A]-X4-G-S (SEQ ID NO: 69), wherein X4 is any amino acid and a junction sequence between SEQ ID NO: 69 and SEQ ID NO: 71 of 4-8 amino acids and wherein the peptide has a length of about 17 to about 35 amino acids, and a heterologous nucleic acid encoding an epimerase radical SAM peptide having an amino acid sequence that has at least 30% identity with Ec ID No. 25 and in which 70% of positions 14-15, 18, 20-24, 27, 58 -60, 63, 83, 84, 87-90, 112, 115, 117, 120, 150, 152, 169, 176, 180, 181, 183, 204, 206-208, 214, 217, 220-225, 228 , 230, 252, 254, 262, 289, 292, 296, and 309 of SEQ ID NO: 25 are preserved and all positions 14-15, 18, 20-24, 27, 58-60, 63, 83 , 84, 87-90, 112, 115, 117, 120, 204, 206-208, 214, 217, 220, 225, 228 and 230 of SEQ ID NO: 25 are conserved and that is capable of co-expressing the peptide and epimerase. 16. The recombinant host cell of claim 15, wherein the peptide comprises a sequence of 24 residues that have at least 40% identity with the sequence at positions 26 to 49 of SEQ ID NO: 1 and that comprises the sequences N/DDLWYF-[V/I/A] (SEQ ID NO: 21) and WI N -X4-GS (SEQ ID NO: 22), wherein X4 is any amino acid, and wherein Peptide is about 25 to about 35 amino acids long. 17. A method for preparing a peptide according to any one of claims 1-9, wherein the method comprises culturing a recombinant host cell according to claim 15 or 16 under conditions suitable for co-expression of the peptide and epimerase and recovering the peptide that has at least two amino acids with a D configuration. 18. Use of a recombinant host cell according to any one of claims 15 and 16 to prepare a peptide according to any one of claims 1-9.